Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports

ABSTRACT

A method of modulation of synthesis capacity on and cleavage properties of synthetic oligomers from solid support is described. The method utilizes linker molecules attached to a solid surface and co-coupling agents that have similar reactivities to the coupling compounds with the surface functional groups. The preferred linker molecules provide an increased density of polymers and more resistance to cleavage from the support surface. The method is particularly useful for synthesis of oligonucleotides, oligonucleotides microarrays, peptides, and peptide microarrays. The stable linkers are also coupled to anchor molecules for synthesis of DNA oligonucleotides using on support purification, eliminating time-consuming chromatography and metal cation presence. Oligonucleotides thus obtained can be directly used for mass analysis, DNA amplification and ligation, hybridization, and many other applications.

This Application is a Continuation Application of U.S. patentapplication Ser. No. 12/493,985 filed Jun. 29, 2009, which is aContinuation Application of U.S. patent application Ser. No. 11/726,269filed Mar. 21, 2007, issued Jun. 30, 2009 as U.S. Pat. No. 7,553,958,which is a Divisional Application of U.S. patent application Ser. No.10/099,382 filed Mar. 13, 2002, issued May 1, 2007 as U.S. Pat. No.7,211,654, which claims priority to expired U.S. Provisional PatentApplication No. 60/275,666 filed Mar. 14, 2001, each of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The instant disclosure pertains to a method for optimization ofsynthesis and purification of synthetic oligomers, such asoligonucleotides and peptides, on a solid support. In particular, thedisclosure pertains to the use of linkers and co-coupling agents forsynthesizing oligonucleotides in a controlled manner and for obtainingoligonucleotides of high quality using simple purification procedures.The method particularly relates to high throughput synthesis ofoligonucleotides for a variety of applications.

BACKGROUND OF THE INVENTION

The growing importance of combinatorial synthesis has created a need fornew resins and linkers having chemical and physical properties toaccommodate a wide range of conditions, since success depends on theability to synthesize diverse sets of molecules on solid supports and tothen cleave those molecules from the supports cleanly and in good yield.

Parallel synthesis, miniaturized analysis and interrogation of librariesof molecules are being perceived as one the most promising approachesavailable to modern chemistry and biology (Gallop et al., (1994) J. Med.Chem. 37, 1233-1251; Gordon et al., (1994) J. Med. Chem. 37, 1385-1401;Ellman et al., (1997) Proc. Natl. Acad. Sci. USA, 94, 2779-2282; Lebl,M. (1999) J. Comb. Chem. 1, 3-24. Examples include applications incombinatorial synthesis and screening of pharmaceutical compounds,biomolecular assays, and gene analysis using oligonucleotide microarraysor DNA chips. A common platform for these micro-chemical and biologicalexperiments is planar surfaces, such as those made from silicon-basedmaterials or synthetic polymers. Among these, glass plates (e.g.microscope slides, which are borosilicate glass) are easily available,easy to handle, and commonly used.

Linkers are molecules that can be attached to a solid support and towhich the desired members of a library of chemical compounds may in turnbe attached. When the construction of the library is complete, thelinker allows clean separation of the target compounds from the solidsupport without harm to the compounds and preferably without damage tothe support. Several linkers have been described in the literature.Their value is constrained by the need to have sufficient stability toallow the steps of combinatorial synthesis under conditions that willnot cleave the linker, while still being cleavable under at least oneset of conditions that is not employed in the synthesis. For example, ifan acid labile linker is employed, then the combinatorial synthesis mustbe restricted to reactions that do not require the presence of an acidof sufficient strength to endanger the integrity of the linker This sortof balancing act often imposes serious constraints on the reactions thatcan be employed in preparing the library.

Accordingly, what needed in the art are improved reagents forfacilitating the synthesis and purification of polymers on solidsupports.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a stable linker [andmore particularly, a selectively cleavable linker, i.e. a linker that iscleavable under at least one set of chemical reaction conditions, whilenot being substantially cleaved (i.e. approximately 90% or greaterremains uncleaved) under another set (or other sets) of reactionconditions] for polymer synthesis comprising a chemical moietyimmobilized on a solid support and not substantially cleaved underpolymer synthesis conditions, which may include chain growth and evenremoval of the protecting groups from the polymer chain. A linker grouptypically has two ends, wherein one of the ends comprises a substrateattaching group and wherein the other of the ends comprises a polymerattaching group, wherein the polymer attaching group is preferablycovalently linked to an anchor moiety and the anchor group has anattaching group for polymer synthesis. The present invention is notlimited to any particular linker group. Indeed, the use of a variety oflinker groups is contemplated, including, but not limited to, alkyl,ether, polyether, alkyl amide groups or a combination of these groups.The present invention is not limited to the use of any particular alkylgroup. Indeed, the use of a variety of alkyl groups is contemplated,including —(CH₂)_(n)—, wherein n is from about 4 to about 20. The use ofa variety of ether and polyether groups is contemplated, including—(OCH₂CH₂)_(n)—, wherein n is from about 1 to about 20. The use of avariety of alkyl amide groups is contemplated, including—(CH₂)_(m)—C(O)NH—(CH₂)_(n) — and —(OCH₂CH₂)_(m)—C(O)NH—(OCH₂CH₂)_(n)—,wherein m and n can be the same or different and m and n are from about1 to about 20. The use of a variety of amide groups having the linkingunits of alkyl or ether bonds is contemplated, including —R₁—C(O)NH—R₂—,wherein R₁ and R₂ are alkyl, ether, and polyether groups.

The present invention is not limited to the use of any particularsubstrate attaching group. Indeed, the use of a variety of substrateattaching groups is contemplated, including, but not limited totrichlorosilyl and trialkyloxysilyl functional groups. The presentinvention is not limited to the use of any particular polymer attachinggroup. Indeed, the use of a variety of polymer attaching groups iscontemplated, including, but not limited to amine, hydroxyl, thiol,carboxylic acid, ester, amide, epoxide, isocyanate, and isothiocyanategroups.

In preferred embodiments of the present invention, the linker iscovalently bound to a support. The present invention is not limited toany particular support. Indeed, the use of a variety of supports iscontemplated, including, but not limited to polymerized LangmuirBlodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄,modified silicon, polyacrylamide, polytetraflouroethylene,polyvinylidendiflouride, polystyrene, polycarbonate, and co-polymers.

The present invention is not limited to the use of any particular anchormoiety. Indeed, the use of a variety of anchor moieties is contemplated,including, but not limited to, those of the following 1,2-diolderivatives of structures shown below:

Wherein P₁ and P₂ are chain units comprised of polymer or linker andpolymer; B is a nucleobase; R₁ are substitution groups, such as CH₃,R₂Ph (R₂ are substitution groups on the phenyl ring, such as SCH₃, Cl,NO₂), CH₂CH₂CN. R is a protecting group, which is OC(O)R₁,t-butyldimethylsilyl (TBDMS), or other protecting groups used for 2′- or3′-O protection of ribonucleotides. Once the protecting group isremoved, the adjacent OH can accelerate the hydrolysis of thephosphodiester bond, resulting in cleavage of the polymer chain.

The present invention is not limited to the use of any particular anchormoiety. Indeed, the use of a variety of anchor moieties is contemplated,including, but not limited to, those of the 2′-deoxyuridine (dU) andabasic moiety of structures shown below:

Wherein P₁ and P₂ are chain units comprised of linker and polymer orpolymer; dU in an oligonucleotide that can be incorporated as itsphosphoramidite and selectively cleaved by uracil-DNA glycosylase (UDG)(from E. coli), which catalyzes the removal of uracil from single- anddouble-stranded DNA. The apyriminic or the abasic sites formed by UNGare susceptible to cleavage by heat under alkaline conditions. Theabasic moiety can be incorporated as its phosphoramidite monomer and islabile under basic conditions; treatment using amines, such aspiperidine, EDA, and N,N′-dimethylethylenediamine causes β- or β- andδ-eliminations to give 5′-phosphate and 3′-phosphate or other3′-products.

The present invention also includes anchor moieties of ribosenucleotides that can be incorporated in regular DNA synthesis usingtheir phosphoramidites. These residues can be cleaved by ribonucleases,such as RNases A (cutting mostly pyrimidines), T₁ (cutting mostly G's)and U₂ (cutting mostly A's). The 3′- and 5′-ends of the cleavedsequences may require further modification using chemical and enzymaticconditions to obtain sequences with 3′- and 5′-fucntional groupsrequired by the subsequent applications. There are many reactionsconditions available for these modifications, including using 5′- or3′-exonucleases for removal of terminal phosphate group.

The present invention is not limited to the use of any particular anchormoiety. Indeed, the use of a variety of anchor moieties is contemplated,including, but not limited to, those of the 2′-deoxyuridine (dU) andabasic moiety of structures shown below:

The present invention is not limited to the use of any particular anchormoiety. Indeed, the use of a variety of anchor moieties is contemplated,including, but not limited to, those of the modified nucleotides ofstructures shown below:

Wherein P₃ and P₄ are chain units comprised of polymer or linker andpolymer; one or both P3 and P4 chains are linked to the nucleotidethrough thioate phosphate (PS) bonds. The PS bond forms in regular DNAor RNA chemical synthesis when the oxidation step employs eithertetraethylthiuram disulfide (TETD) or 3H-1,2-bensodithiol-3-one1,1-dioxide (BDTD) for sulfurizing phosphite trimesters formed fromcoupling of phosphoramidites (Spitzer, S.; Eckstein, F. (1988) NucleicAcids. Res. 16, 11691-11704). The PS linkage can be selectively cleavedby the addition of I₂ (Strobel, S. A., and Shetty, K. Proc. Natl. Acad.Sci. USA. 94, 2903-2908).

In preferred embodiments, the anchor moiety is stable under conditionsused for polymer synthesis, which may include conditions for chaingrowth as well as conditions for removal of the protecting groups fromthe polymer chain. The anchor moieties of the present invention may becleavable under certain selected conditions. The present invention isnot limited to any particular set of selective cleavage conditions.Indeed, the present invention contemplates that a variety of cleavageconditions may be utilized when appropriate, including 2-OH assisted1-phosphate hydrolysis and enzymatic cleavage of the chemical bonds. Inother embodiments of the present invention, the anchor moiety includes apolymer attaching group. In still further embodiments, a polymer isattached to the anchor moiety. The present invention is not limited toany particular polymer. Indeed, a variety of polymers are contemplated,including, but not limited to peptides and oligonucleotides.

The present invention is not limited to the use of any particular anchormoiety. Indeed, the use of a variety of anchor moieties is contemplated,including, but not limited to, those of the following structure:

wherein B is a purine or pyrimidine base, R is H or CH₃ and R₄ is apolymer.

In still other embodiments, the present invention provides compoundspossessing the structure:Rs-L-Rpwherein R_(s) is a substrate attaching group, R_(p) is a polymerattaching group, and L is the linker.

The present invention is not limited to the use of any particularsubstrate attaching group (R_(s)). Indeed, the use of a variety ofsubstrate attaching groups is contemplated, including, but not limitedto chlorosilyl and alkyloxysilyl functional groups. The presentinvention is not limited to the use of any particular polymer attachinggroup. Indeed, the use of a variety of polymer attaching groups iscontemplated, including, but not limited to amine, hydroxyl, thiol,carboxylic acid, ester, amide, epoxide, isocyanate, and isothiocyanategroups.

In some embodiments, R_(p) is selected from the group including, but notlimited to amine, hydroxyl, thiol, carboxylic acid, ester, amide,epoxide, isocyanate, and isothiocyanate groups.

In some embodiments of the present invention, the linker is covalentlybound to a support. The present invention is not limited to anyparticular support. Indeed, the use of a variety of supports iscontemplated, including, but not limited to polymerized LangmuirBlodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄,modified silicon, polyacrylamide, polytetraflouroethylene,polyvinylidendiflouride, polystyrene, and polycarbonate.

In still further embodiments, the present invention provides methods forsynthesizing oligonucleotides comprising: providing a substrate; aplurality of stable linkers; a plurality of anchor moieties; andnucleotide monomers; derivitizing the substrate with the plurality ofstable linkers; attaching the anchor moieties to the stable linkers; andsynthesizing oligonucleotides on the plurality of anchor moieties. Insome embodiments, the methods further comprise deprotecting theoligonucleotides and selectively cleaving the oligonucleotides from thesubstrate by reacting the substrate under conditions such that thepolymer is cleaved at the anchor moiety.

In still further embodiments, the present invention provides methods forcontrolling the number of oligonucleotides synthesized at apredetermined site on a substrate comprising: providing a substrate; aplurality of stable linkers; a plurality of anchor moieties; nucleotidemonomers; and co-coupling agents; derivitizing the substrate with saidplurality of stable linkers; attaching the anchor moieties to the stablelinkers; and synthesizing a oligonucleotide on the plurality of anchormoieties from the monomers in the presence of the co-coupling agentsunder conditions such that at least a portion of the oligonucleotidesare terminated.

In still other embodiments, the present invention provides methods ofpurifying oligonucleotides comprising: providing: a substrate comprisingoligonucleotides attached to a substrate via an anchor moiety attachedto a stable linker group, a deprotecting solution, and a wash solution;deprotecting said oligonucleotides with said deprotecting solution,washing said oligonucleotides attached to a substrate with said washsolution, and cleaving said oligonucleotides at said anchor group toprovide purified oligonucleotides, wherein said purifiedoligonucleotides are characterized by the substantial absence of metalions and/or other contaminants and said stable linker group remainsattached to said substrate.

In still other embodiments, the present invention provides methods ofobtaining purified oligonucleotides comprising: providing: a substratecomprising oligonucleotides attached to a substrate via an anchor moietyattached to a stable linker group, a deprotecting solution, a washsolution, and a cleavage solution; deprotecting said oligonucleotideswith said deprotecting solution, washing said oligonucleotides attachedto a substrate with said wash solution, and cleaving saidoligonucleotide using said cleavage solution at said anchor group toprovide purified oligonucleotides, wherein said purifiedoligonucleotides are characterized by the substantial absence of metalions and said stable linker group remains attached to said substrate.The oligonucleotides thus obtained have many applications, such assubstrates of nucleases, polymerases, kinases, or ligases, known tothose of skilled in the art.

DESCRIPTION OF THE FIGURES

FIG. 1 provides examples of the chemical structure of the linker groupsof the present invention attached to a solid substrate.

FIG. 2 provides exemplary chemical structures for a fluorescein tag, achain terminator (co-coupling agent) and an anchor.

FIG. 3 displays electrophoresis gel profiles of T₁₀ cleaved from glassplates at 15, 30 and 60 min upon treatment with conc. aq. NH₄OH. The T₁₀with amide linker is shown on the left panel and the T₁₀ with C₈ linkeris shown on the right panel.

FIG. 4 presents results of an assay of oligonucleotide synthesis using atermination nucleophosphoramidite, 5′-MeO-T, to probe the presence ofavailable sites for coupling with a phosphoramidite at differentreaction stages. (A) Regular T₃ synthesis on glass plates. (B)Illustration of the use of termination monomer. T on glass plate iscoupled with MeO-T, resulting in the formation of a terminated dimerT-T(OMe), which can not undergo further chain growth. (C) Illustrationof the hypothesis for reaction with more hindered surface sites inseveral continued reaction cycles. (D) ³²P-gel electrophoresis analysisof the experiments using the termination 5′-MeO-T at different stages ofoligonucleotide synthesis.

FIG. 5 presents a comparison of the probe sequences synthesized usingthe amide and C8 linkers and used for three time hybridizationexperiments.

FIG. 6 presents a schematic depiction of the synthesis of an exemplaryanchor moiety.

FIG. 7 presents a schematic depiction of the synthesis of an exemplaryoligonucleotide.

FIG. 8 presents a schematic depiction of deprotection and cleavage of anexemplary oligonucleotide.

FIG. 9 presents HPLC data for oligonucleotides (SEQ ID NOs: 5 and 7)synthesized on the supports of the present invention.

FIG. 10 presents HPLC data for the enzyme purified sequence cleaved fromCPG.

FIG. 11 presents the results of PCR experiments conducted witholigonucleotide primers synthesized on the supports of the presentinvention.

DEFINITIONS

The following terms are intended to have the following general meaningas they are used herein:

The term “substrates” and “solid supports” are used interchangeably torefer to any material that is suitable for derivatization with a linkergroup. Examples of substrates include, but are not limited to glass,Si-based materials, functionalized polystyrene, functionalizedpolyethyleneglycol, functionalized organic polymers, nitrocellulose ornylon membranes, paper, cotton, and materials suitable for synthesis.Solid supports need not be flat. Supports include any type of shapeincluding spherical shapes (e.g., beads). Materials attached to solidsupport may be attached to any portion of the solid support (e.g., maybe attached to an interior portion of a porous solid support material).Preferred embodiments of the present invention have biological moleculessuch as oligonucleotides and peptides attached to solid supports. Acompound is “attached” to a solid support when it is associated with thesolid support through a non-random chemical or physical interaction. Insome preferred embodiments, the attachment is through a covalent bond.

As used herein, the terms “linker” and “linker group” are usedinterchangeably to refer to chemical moieties that are attachable to asolid support on one end and an anchor group or polymer on the otherend. The “linker” and “linker group” are atoms or molecules that link orbond two entities (e.g., solid supports, oligonucleotides, or othermolecules), but that is not a part of either of the individual linkedentities. In general, linker molecules are oligomeric chain moietiescontaining 1-200 linearly connected chemical bonds. One end of a linkerchain is immobilized on substrate surface, such as through —SiO— bondformation. The other end of a linker chain contains a functional groupthat can be converted to an OH or an NH₂ group. Examples of linkersinclude, but are not limited to the chemical moieties shown in FIG. 1and —(OCH₂CH₂)_(n)—, wherein n is from about 1 to about 20. The use of avariety of alkyl amide groups is contemplated, including—(CH₂)_(m)—C(O)NH—(CH₂)_(n)— and —(OCH₂CH₂)_(m)—C(O)NH—(OCH₂CH₂)_(n)—,wherein m and n can be the same or different and m and n are from about1 to about 20. The use of a variety of amide groups having the linkingunits of alkyl or ether bonds is contemplated, including —R₁—C(O)NH—R₂—,wherein R₁ and R₂ are alkyl, ether, and polyether groups. Linkers canhave substitutions to have branched chain structures, such as dendriticstructures. Multiple linkers can be covalently connected to form anextended linker chain.

The term “anchor group or moiety” refers to a chemical moiety thatconnects a linker and a synthesized oligonucleotide or other polymer andwhich can be selectively cleaved to release oligonucleotides or otherpolymers from substrate surface. For example, the anchor may include thestructure —C(X)—C(Y)— (X may be OPO₂O-oligonucleotide), (Y is afunctional group that may function as a nucleophile, for example, Y maybe an OH, NH₂ or SH). Preferably, the —C(X)—C(Y)— is part of a ringmoiety and further a five member ring moiety. The anchor may include dU,abasic group, ribonucleotides, thioate phosphodiester, when incorporatedinto oligonucleotides, which can selectively cleaved by treatment withspecific enzymatic digestion or chemical degradation conditions.

The term “protected nucleotides” refers to nucleotides containingnucleobase protecting groups, such as 4-NH-benzol in cytidine andadenine and 2-NH-isobutyryl in guanosine, sugar protecting groups, suchas 2′-O-t-butyldimethylsilyl in ribonucleotides, and phosphateprotecting groups, such as P—O-(2-cyano)ethylphosphine, etc. “Protectinggroup” refers to a molecule or chemical group that is covalentlyattached to a moiety of a compound to prevent chemical modification ofthe moiety of the compound or modification of specific chemical groupsof the compound. For example, protecting groups may be attached to areactive group of a compound to prevent the reactive group fromparticipating in chemical reactions including, for example,intramolecular reactions. In some cases, a protecting group may act as aleaving group, such that when the molecule is added to another compoundin a desired synthesis reaction, the protecting group is lost, allowinga reactive group to participate in covalent bonding to the compound. Thephosphoramidites of the present invention typically contain one or moreprotective groups prior to their addition to nucleic acid molecules. Forexample, the reactive phosphate of the phosphoramidite (i.e., thephosphate group that is covalently attached to another molecule when thephosphoramidite is added to the other molecule) may contain one or moreprotecting groups. A detailed description of phosphoramidites and theiraddition to nucleic acid molecules is provided Beaucage and Iyer(Tetrahedron 49:1925 [1993]), herein incorporated by reference in itsentirety.

As used herein, the term “stable”, when used in reference to a linker oran anchor group, refers to a property of the compound or the chemicalmoiety which is not cleaved by certain reactions conditions, butselectively cleavable by different reaction conditions. These orthogonalreactions are well established in solid phase synthesis. The presentinvention is not limited to any particular set of selective cleavageconditions. Indeed, the present invention contemplates that the siloxanelinkers are stable under anhydrous ethylene diamine treatment, but avariety of cleavage conditions may be utilized when appropriate,including base hydrolysis of the Si—O bond. Further, the presentinvention contemplates that the 1,2-diol anchors are stable to basichydrolysis when one of the OH group is protected with a protectingmoiety, but a variety of cleavage conditions may be utilized after theOH protecting group is removed, including 2-OH assisted 1-phosphatehydrolysis under basic conditions. Thus, the present inventioncontemplates in one embodiment linkers and/or anchor groups that arestable to basic hydrolysis. In another embodiment, the present inventioncontemplates linkers and/or anchor groups that are stable to acidhydrolysis.

As used herein, the term “selective cleavable”, when used in referenceto a linker or an anchor group, refers to a property of the compound orthe chemical moiety is not cleaved by certain reactions conditions, butselectively cleavable by different reaction conditions. These orthogonalreactions are well established in solid phase synthesis. The presentinvention is not limited to any particular set of selective cleavageconditions. Indeed, the present invention contemplates that the siloxanelinkers are stable under anhydrous ethylene diamine treatment, but avariety of cleavage conditions may be utilized when appropriate,including base hydrolysis of the Si—O bond. Further, the presentinvention contemplates that the 1,2-diol anchors are stable to basichydrolysis when one of the OH group is protected with a protectingmoiety, but a variety of cleavage conditions may be utilized after theOH protecting group is removed, including 2-OH assisted 1-phosphatehydrolysis under basic conditions. Further, the present inventioncontemplates that dU, abasic moiety, ribonucleotides, and thioatephosphodiester are stable under regular DNA or RNA synthesis conditionsbut may be selectively cleaved by specific chemical or enzymatictreatments.

As used herein, the term “substrate attaching group” refers to anychemical group that is useful for attaching a linker to a substrate.Examples of substrate attaching groups include, but are not limited to,monochlorosilyl, monoalkoxysilyl, trichlorosilyl or trialkoxysilylgroups.

As used herein, the term “polymer attaching group” refers to afunctional group or groups that can be converted to a functional group,for example, an OH or an NH₂ group, that is used for initiatingsynthesis of a polymer on a linker or attaching an anchor moiety to alinker. Examples of polymer attaching groups include, but are notlimited to, amino, hydroxy, thiol, carboxylic acid, ester, amide,isocyanate or isothiocyanate group, most preferably an OH or a NH₂group. Methods for such functionalization are well known in the art(See, e.g., Bigley et al., J. Chem. Soc. (B):1811-18 (1970).

As used herein, the term “synthesis initiation site” refers to achemical group on a linker or an anchor moiety or any other chemicalentity that is used as a site for initiating synthesis of a polymerchain.

As used herein, the term “spacer” refers to a chemical group connectedto a linker or an anchor moiety that is used to extend the length of thelinker moiety and as a site for initiating synthesis of a polymer chain.Examples of spacer include, but are not limited to, ethyleneglycolpolymer, alkyl, oligonucleotides, peptides, peptditomimetics.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more deoxyribonucleotides or ribonucleotides,preferably at least 4 nucleotides, more preferably at least about 10-15nucleotides and more preferably at least about 15 to 200 nucleotides.The exact size will depend on many factors, which in turn depend on theultimate function or use of the oligonucleotide. The oligonucleotide maybe generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, PCR, ligation, or a combinationthereof.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction. Anoligonucleotide sequence is written in 5′- to 3′ direction byconvention.

As used herein, the term “co-coupling agent” refers to a compound whichwhen incorporated into a polymer serves as chain terminator, i.e.terminating the chain growth. The co-coupling agent preferably has astructure similar to the monomers used in the polymer synthesisreaction. The co-coupling agent can be mixed with coupling agent in thesynthesis, resulting in a mixture of extendible and non-extendiblesequences that no longer can be extended and sequences that cancontinuously grow in length.

As used here in the term “coupling agent or monomer” refers to abuilding block in polymer synthesis. The compound has a reactive groupwhich reacts with functional groups of the reacting compounds on solidsurface and has a protected reactive group which in a later synthesisstep can be deprotected to form reactive functional group for furtherreaction with another coupling agent or monomer.

As used here in the term “terminator or chain terminator” refers to acompound which does not contain the same protected reactive sites as thecoupling agent. Therefore, when included in an appropriate ratio with acoupling agent or monomer, terminator forms a number of inactivesequences that cannot be extended in further reactions. Examples ofterminator or chain terminator useful in the present invention includenucleophosphoramidites and nucleophosphonates that cannot be extended,for example 5′-MeO-T.

As used herein, the terms “complementary” or “complementarity” are usedin reference to oligonucleotides (i.e., a sequence of nucleotides suchas an oligonucleotide or a target nucleic acid) related by thebase-pairing rules. For example, for the sequence “5′-A-G-T-3′,” iscomplementary to the sequence “3′-T-C-A-S′.” Complementarity may be“partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods which depend upon binding between nucleicacids. Either term may also be used in reference to individualnucleotides, especially within the context of oligonucleotides. Forexample, a particular nucleotide within an oligonucleotide may be notedfor its complementarity, or lack thereof, to a nucleotide within anothernucleic acid strand, in contrast or comparison to the complementaritybetween the rest of the oligonucleotide and the nucleic acid strand.

The term “homology” and “homologous” refers to a degree of identity ofat least two compounds or sequences. There may be partial homology orcomplete homology. A partially homologous sequence is one that is lessthan 100% identical to another sequence.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, and the thermodynamics of the formed hybrid. “Hybridization”methods involve the annealing of one nucleic acid to another,complementary nucleic acid, i.e., a nucleic acid having a complementarynucleotide sequence. The ability of two polymers of nucleic acidcontaining complementary sequences to find each other and anneal throughbase pairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modern biology.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanineComplementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences and sufficient hybridization stability.Thus, conditions of “weak” or “low” stringency are often required whenit is desired that nucleic acids which are not completely complementaryto one anotheror have lower hybridization stability be hybridized orannealed together.

The term “label” as used herein refers to any atom or molecule that canbe used to provide a detectable (preferably quantifiable) signal, andthat can be attached to a nucleic acid or protein or other polymers.Labels may provide signals detectable by fluorescence, radioactivity,colorimetry, gravimetry, X-ray diffraction or absorption, magnetism,enzymatic activity, and the like. A label may be a charged moiety(positive or negative charge) or alternatively, may be charge neutral.Labels can include or consist of nucleic acid or protein sequence, solong as the sequence comprising the label is detectable.

As used herein, the term “dye” refers to a molecule, compound, orsubstance that can provide an optically detectable signal (e.g.,fluorescent, luminescent, colorimetric, etc). For example, dyes includefluorescent molecules that can be associated with nucleic acid molecules(e.g., Cy3).

As used herein, the term “directly bonded,” in reference to two or moremolecules refers to covalent bonding between them without anyintervening linking group or spacer groups that are not part of parentmolecules.

As used herein, the term “purified” or “to purify” refers to the removalof contaminants, such as metal ions, from a sample of the desiredsynthesized polymer. In like manner, the terms “purified” or “to purify”may also refers to enrichment of the desired synthesized polymerrelative to other components in a sample. For example, the presentinvention contemplates purification wheein the desired synthesizedpolymer is present in amounts that represent 50% or greater of thecomponents in a sample (and more preferably, 70% or greater; still morepreferably 80% or greater; and most preferably greater than 90%).Removal of contaminants or enrichment of the desired synthesized polymercan refer to samples that have the desired polymer attached to a supportor samples in which the desired polymer has been cleaved from thesupport. In one embodiment, the present invention contemplatespurification wherein greater than 90% of contaminants have been removed.

DESCRIPTION OF THE INVENTION

A variety of synthetic approaches have been developed for preparation ofoligonucleotide sequences. Typically, oligonucleotides are synthesizedutilizing a building block approach which involves the sequentialaddition of nucleotides onto a growing oligonucleotide chain immobilizedon to a solid support. Because every DNA oligonucleotide may have any of4 different initial nucleotides, it is necessary to maintain a supply of4 different nucleoside (A, C, G and T) loaded solid supports to be ableto synthesize any given DNA sequence. In the case of DNA synthesis, thefirst nucleoside from the 3′ end of the DNA sequence is typicallypreloaded on the solid support through an ester linkage. For example, ifthe sequence that is to be synthesized contains a T nucleoside at the 3′end, a T support is employed and the balance of the nucleotides in theDNA sequence added thereto (e.g., using an automated DNA synthesizer).At the end of the total DNA synthesis, the oligonucleotide is cleavedfrom the solid support through the hydrolysis of the ester linkage.Taking into consideration RNA synthesis procedures, an additional4-different nucleoside loaded solid supports must be available to theuser. Similar considerations apply if any specialty modified nucleosideis desired at the 3′ end.

Maintaining a supply of at least 8 different prederivatized solidsupports is inconvenient and expensive. An additional consideration isthe relatively short shelf life of nucleoside derivatized solidsupports. Typically, after one year storage such solid supports are notlonger usable. There is also the possibility that synthetic proceduresmay be initiated mistakenly with the wrong support leading to disastrousconsequences in the final applications of the oligonucleotides.

In order to alleviate these problems some researchers have pursueddeveloping some type of universal solid support. For example, deBear etal. derivatized glass supports with 2′ (3′)-O-benzoyluridine5′-O-succinyl so that the uridine moiety is linked to the glass via anester (succinate) linkage. [de Bear et al., Nucleosides and Nucleotides6, 821-830 (1987)]. Oligonucleotide synthesis takes place by addingnucleotide monomers to the 2′ or 3′ position of the uridine. Followingthe synthesis, the new oligonucleotides can be released from the glass,deprotected and cleaved from the uridylyl terminus in one reaction. Theuridyl functionality is cleaved from the solid support in this cleavingreaction.

Crea and Horn suggested a similar approach which involved preparing thedimer5′-O-p-chlorophenylphospho-2′(3′)-O-acetyluridilyl-[2′(3′)-3′]-5′-O-dimethoxytritylthymidinep-chlorophenylester and attaching the dimer to cellulose via a phosphatelinkage. [R. Crea & T. Horn, Nucleic Acids Research 8, 2331 (1980)]. The5′ position of the thymidine is available for oligonucleotide attachmentand synthesis. The subsequent use of aqueous concentrated ammoniaresults in the release of the synthesized oligonucleotide from thecellulose leaving the uridine portion of the dimer attached to thecellulose. Although Crea and Horn utilized the reactive vicinal OHgroups on the uridine as the release site for the oligonucleotide fromthe uridine, the solid support suggested in this reference is not auniversal solid support since the initial oligonucleotide isincorporated in the solid support reagent and a different support isrequired for oligonucleotides incorporating a different firstnucleoside.

More recently, Schwartz et al. attached an adapter,2′(3′)-O-dimethoxytrityl-3′(2′)-O-benzoyluridine-5′-O-2-cyanoethylN,N-diisopropylphosphoramidite, to a thymidine derivatized polystyreneand synthesized an oligonucleotide from the O-dimethoxytrityl (O-DMT)position of the uridine after removal of the DMT group (M. E. Schwartz,R. R. Breaker, G. T. Asteriadis, and G. R. Gough, Tetrahedron Letters,Vol. 36, No. 1, pp 27-30, 1995). While this approach provides auniversal solid support for oligonucleotide synthesis, the cleaving stepreleases the adapter and the thymidine from the support and then cleavesthe synthesized oligonucleotide from the uridine. Thus, the purificationprocess requires removing the thymidine linker and the cleavingprocesses.

The aforementioned solid supports and methods for their use have severaldisadvantages in terms of the convenience and efficiency of thesubsequent oligonucleotide cleaving steps. When ammonia which has beenwidely accepted as a safe reagent for DNA synthesis is utilized forcleaving, as taught by deBear et al., the cleavage time is as long as 24hours at 65° C. In view of the growing trend to produce oligonucleotidesas quickly as possible, this is an unacceptably long period of time.Decreasing the time required for cleaving the uridylyl from anoligonucleotide at the uridine 3′ position typically uses Pb²⁺ or Mg²⁺ion catalyst system or the action of strong alkali hydroxides.Necessarily these processes require a separate isolation step to removethe ion used. Additionally, when strong alkali bases are used in thecleaving processes, considerable side reactions in the form of cytosinedeamination occur.

U.S. Pat. No. 5,919,523 (Affymetrix; incorporated herein by reference)describes derivatization of solid supports and methods for oligomersynthesis. The methods provide polymer-coated support for use insolid-phase synthesis (polyethyleneimine, polyethyleneglycol, polyvinylalcohol, etc.). The polymer coating may be functionalized to containsynthesis initiation sites. The method also describes reducing surfacedensity of functional groups using protected amino acids to react withfunctional groups on polymer coating.

PCT publication WO046231 (Amersham; incorporated herein by reference)describes a method for purifying an oligonucleotide that comprisesproviding an oligonucleotide attached to a substrate, wherein theoligonucleotide contains phosphate protecting groups; contacting theoligonucleotide with a reagent, e.g., an organic amine, that cleaves thephosphate protecting groups from the oligonucleotide without detachingthe oligonucleotide from the substrate; isolating the oligonucleotideattached to the substrate from the cleaved phosphate protecting groups;and cleaving the oligonucleotide from the substrate. The side reactionsinvolving acrylonitrile (formed from deprotection of phosphate) andnucleotides can be avoided. This method provides crude oligonucleotidemixtures that are easier to purify and from which the desiredfull-length oligonucleotide product. Linkers used are those on standardCPG containing a succinyl linkage.

U.S. Pat. No. 5,738,829 (T. Kempe; incorporated herein by reference)describes an apparatus connected to a DNA synthesizer for gas phasedeprotection of oligonucleotides that are covalently bound to solidsupport using ammonia or ammonium hydroxide vapors.

U.S. Pat. No. 5,656,741 (Chow, F. and Kempe, T., incorporated herein byreference) describe a process for the cleavage, deprotection, andrecovery of a synthetic oligonucleotide by immersing the support in abasic solution, whereby cleavage occurred first and followed bydeprotection. The cleaved and deprotected oligonucleotide was recoveredby precipitation from solution.

U.S. Pat. No. 5,750,672 (Kempe, T., incorporated herein by reference)describes a method for recovering synthesized oligonucleotides from asolid support that includes the step of incubating the solid supportwith an anhydrous amine reagent under conditions suitable to cleave anddeprotect the oligonucleotide. The cleaved and deprotectedoligonucleotide will be substantially insoluble in the reagent and/orwill exhibit preferential affinity for the support. Reagent kits for usein such a method and cleaved, deprotected oligonucleotides prepared bymeans of such a method are provided.

U.S. Pat. No. 5,869,696 (Beckman Instruments; incorporated herein byreference) describes universal solid support oligonucleotide synthesisreagents, oligonucleotide synthesis processes, and reagents for cleavingoligonucleotides from solid supports. Oligonucleotide synthesis on solidsupport is through a ring moiety having vicinal groups that can attackthe other when one of the two is not protected, causing cleavage ofoligonucleotide synthesized. The linkage between the ring moiety and thesupport is not stable to the cleavage condition. The universal supportis intended to reduce the number of the types of support needed forconventional oligonucleotide synthesis on cleavable linkers.

U.S. Pat. No. 6,090,934 (Kumar, P. and Gupta, K. C., incorporated hereinby reference) describes a universal polymer support containing anorganic aliphatic molecule of structure having a least a pair ofcis-hydroxyl groups where on of the hydroxyl groups is attached to thepolymer support through a covalent linkage and the other hydroxyl groupis protected by an acid labile group, which is activated foroligonucleotide synthesis. Upon completion of the synthesis, theoligonucleotide on solid support is treated with a basic solution. Thecleavage of the linkage between the aliphatic molecule and the polymersupport frees a hydroxyl, which in turn attacks the adjacent phosphategroup to form cyclic phosphate and give free oligonucleotide.

U.S. Pat. No. 6,015,895 (Pon, R. T. and Yu, S, incorporated herein byreference) describes a process for producing a chemically modified solidsupport for oligonucleotide synthesis, the process comprising the stepsof reacting a linker compound, which is a substituted or unsubstitutedC1-C20 alkyl group, a substituted or unsubstituted C5-C30 aryl group anda substituted or unsubstituted C5-C40 alkaryl group, with an OH of adesired nucleoside to produce a derivatized nucleoside having an esterlinkage; and a solid support capable of entering into an esterificationreaction, to produce the linker arm.

U.S. Pat. No. 6,043,353 (Pon, R. T. and Yu, S, incorporated herein byreference) describes reusable solid support having linkers consisting ofa substituted or unsubstituted C1-C20 alkyl group, C5-C30 aryl group, orC5-C40 alkylaryl group. The linker groups have a stable portion linkedto another portion through a base cleavable bond, such as an ester bond.The stable portion of the linker can be reused after each cleavage ofoligonucleotides from support.

The present invention provides improved systems for synthesizingpolymers on solid supports. In particular, the present inventionprovides linker systems that provide an increased density of reactionsites on solid supports. In one embodiment, the present inventionachieves a density such that the linkers are at least two times (andmore preferably at least four times) more densely packed (e.g., on asurface) than conventional linkers. These linker systems improvestability of linkers and the linker-polymer connectivity under normalpolymer reaction conditions, such as in amine solutions. The presentinvention is not limited to any particular mechanism. Indeed, anunderstanding of the mechanism is not required to practice the presentinvention. Nevertheless, it is believed that the increased packingdensity of the linker systems of the present invention provide both amore ordered surface and increased resistance to cleavage as compared toconventional linker systems. In preferred embodiments, the stablelinkers are modified to include a cleavable anchor group at the end ofthe linker opposite of the solid support. The polymer (e.g.,polynucleotide or polyamino acid) is then synthesized from a startingpoint (e.g., a functional group) on the anchor molecule. Followingsynthesis of the polymer, the protecting groups on the polymer can beremoved, the solid support can be conveniently washed and then treatedunder appropriate conditions so that the anchor group is cleaved,releasing the washed polymer from the support. The density of thepolymers synthesized on the solid support can be conveniently controlledby including co-coupling agents that are structurally similar to themonomers of the polymer being synthesized. The co-coupling agentsterminate polymer synthesis at sites where they incorporated, therebydecreasing the number of polymer chains synthesized. The polymersynthesized can be applied to various applications. Reagents and methodsprovided by the present invention are described below in the followingsections:

I. Solid Supports

It is contemplated that the reagents and methods of the presentinvention may be utilized with a variety of solid supports. In general,any solid support that may be derivatized with the linker groups (SeeSection II) of the present invention finds use in the present invention.Accordingly, the present invention is not limited to the use of any onesolid support.

In particular, the solid substrate may be biological, nonbiological,organic, inorganic, or a combination of any of these, existing asparticles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides, etc. Thesolid substrate is preferably flat but may take on alternative surfaceconfigurations. For example, the solid substrate may contain raised ordepressed regions on which synthesis takes place. In some embodiments,the solid substrate will be chosen to provide appropriatelight-absorbing characteristics. For example, the substrate may be apolymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs,GaP, SiO₂, SiN₄, modified silicon, nitrocellulose and nylon membranes,or any one of a variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene,polycarbonate, or combinations thereof. Other suitable solid substratematerials will be readily apparent to those of skill in the art.Preferably, the surface of the solid substrate will contain reactivegroups, which could be carboxyl, amino, hydroxyl, thiol, or the like.More preferably, the surface will be optically transparent and will havesurface Si—OH functionalities, such as those found on silica surfaces.

II. Linker Groups

A linker group typically has two ends, wherein one of the ends comprisesa substrate attaching group and wherein the other of the ends comprisesa polymer attaching group, wherein the polymer attaching group ispreferably covalently linked to an anchor moiety and the anchor grouphas an attaching group for polymer synthesis. The present invention isnot limited to any particular linker group. Indeed, the use of a varietyof linker groups is contemplated, including, but not limited to, alkyl,ether, polyether, alkyl amide groups or a combination of these groups.The present invention is not limited to the use of any particular alkylgroup. Indeed, the use of a variety of alkyl groups is contemplated,including —(CH₂)_(n)—, wherein n is from about 4 to about 20. The use ofa variety of ether and polyether groups is contemplated, including—(OCH₂CH₂)_(n)—, wherein n is from about 1 to about 20. The use of avariety of alkyl amide groups is contemplated, including—(CH₂)_(m)—C(O)NH—(CH₂)_(n)— and —(OCH₂CH₂)_(m)—C(O)NH—(OCH₂CH₂)_(n)—,wherein m and n can be the same or different and m and n are from about1 to about 20. The use of a variety of amide groups having the linkingunits of alkyl or ether bonds is contemplated, including —R₁—C(O)NH—R₂—,wherein R₁ and R₂ are alkyl, ether, and polyether groups. The linkerscan be terminated with a functional group, such as OH, SH, NHR (R═H orsubstitution group, such as CH₃, CH₂CH₃, Ph), aldehyde, carboxalic acid,ester, or other typical reactive groups. The linkers can also connect toan anchor group or a polymer. Multiple linkers can be covalentlyconnected to form an extended linker chain.

The present invention is not limited to the use of any particularsubstrate attaching group. Indeed, the use of a variety of substrateattaching groups is contemplated, including, but not limited tochlorosilyl, alkyloxysilyl, alkylchlorosily, and alkylalkoxysilyfunctional groups. The present invention is not limited to the use ofany particular polymer attaching group. Indeed, the use of a variety ofpolymer attaching groups is contemplated, including, but not limited toamine, hydroxyl, thiol, carboxylic acid, ester, amide, epoxide,isocyanate, and isothiocyanate groups.

In preferred embodiments, a silicon-containing substrate isfunctionalized with a hot pirhana solution (e.g., concentratedH₂SO₄:H₂O₂, 50:50 v/v) for a short period of time (e.g., 15 min). The—(CH₂)₆CHCH₂ linker that includes a silane functionality as part of itssubstrate attaching group is then reacted with the functionalizedsubstrate to provide a substrate or solid support derivatized with alinker group. The polymer attaching group is then functionalized bytreatment with a suitable functionalizing agent (e.g., BH₃/THF/H₂O₂,BH₃/NaOAc, BH₃/NaOH, or BH₃/NaOH).

The present invention is not limited to any particular mechanism.Indeed, an understanding of the mechanism is not required to make andthe invention. Nevertheless, the use of the linkers described aboveprovides a derivatized surface comprising a higher density of thelinking groups. It is contemplated that the high density of linkinggroups results in higher yield of the total sequences synthesized onsolid surfaces and may increase the resistance to surface cleavageduring normal polymer synthesis steps such as activation anddeprotection of phosphoramidites. It is contemplated that the increasedsequence density due to different linkers used is highly desirable forthe subsequent applications of the polymers, such as oligonucleotidesand peptides. The resistance to cleavage will allow the multiple usageof biochips, thereby greatly reducing the costs associated with suchchips.

The increased density of the linking groups of the present invention canbe assayed by loading of controlled porous glass (CPG). In preferredembodiments, the linking groups of the present invention are capable ofa loading density on CPG (>500 Å pore size) of about greater than 10μmol/g of CPG, preferably greater than 20 μmol/g of CPG, and mostpreferably greater than about 100 μmol/g of CPG.

III. Anchor Groups

The present invention also provides anchors groups or moieties forattachment to the linker through the polymer attachment group. Inpreferred embodiments, the anchor group includes a reactive site forattachment to the polymer attachment site of the linker. In furtherpreferred embodiments, the anchor group includes a synthesis initiationsite from which a polymer can be synthesized. In still further preferredembodiments, the anchor is selectively cleavable, preferably not beingcleaved by regular synthesis, including coupling and deprotecting steps.

In particular, in some embodiments, the anchor groups of the presentinvention are organic aliphatic molecules (e.g., butane-2,3-diol,1,2,3-trihydroxyheptane, 1,2,3-hexanetriol and the like) of thefollowing general structure:

where R₁═H—(CH₂)_(n)— and R₂═—CH₂—OH, —(CH₂)_(n)—H; n=1-4; andDMTr=4,4′-dimethoxytrityl. Those skilled in the art will recognize thatother protecting groups may be utilized in place of DMT.

The present invention is not limited to the use of any particular anchormoiety. Indeed, the use of a variety of anchor moieties is contemplated,including, but not limited to, those of the following 1,2-diolderivatives of structures shown below:

Wherein P₁ and P₂ are chain units comprised of linker and polymer orpolymer; B is a nucleobase; R₁ are substitution groups, such as CH₃,R₂Ph (R₂ are substitution groups on the phenyl ring, such as SCH₃, Cl,NO₂), CH₂CH₂CN. R is a protecting group, which is OC(O)R₁,t-butyldimethylsilyl (TBDMS), or other protecting groups used for 2′- or3′-O protection of ribonucleotides. Once the protecting group isremoved, the adjacent OH can accelerate the hydrolysis of thephosphordiester bond, resulting in cleavage of the polymer chain.

The diol compounds can be treated with one equivalent of ahomobifunctional alkanoic acid halide (e.g., oxalyl chloride, succinoylchloride, adipoyl chloride and the like) and reacted with the polymerattachment group which has hydroxyl or aminoalkyl functionalities. Theunreacted functional groups in the above diol derivatives then can thenbe capped with dry alkanol (e.g., MeOH, EtOH, propanol and the like) forblocking the residual functional groups followed by washing with dryalkanol and dialkyl ether, respectively.

In other embodiments of the present invention, the anchor molecule hasthe following general structure:Rs-L-Rpwherein L is a linker group (which may in turn be covalently bound to asolid substrate as described above); R_(s) is the surface attachinggroup; R_(p) is the polymer attaching group and it is

or an ether containing group (e.g., polyethylene glycol), where R₃ ishydrogen or alkyl and R₄ is a phosphate protecting group; and R_(b) is aring moiety having vicinal groups —XR₁ and —YR₂ wherein each of X and Yis independently selected from the group consisting of O, S and NH andone of R₁ and R₂ is a blocking moiety and the other is hydrogen or ahydroxy protecting group suitable for protecting OH, SH, or NH₂.Recognizing that when R_(p) is a phosphoramidite or its oxidized formphosphoramidate, those skilled in the art will appreciate that R₃ ispreferably hydrogen. This is because these oligonucleotide synthesisreagents are generally prepared using a primary amine. However, thoseskilled in the art will also appreciate that R₃ can be alkyl because thephosphoramidate can be prepared using secondary amines. Phosphateprotecting group R₄ is suitably any group capable of protecting thephosphorous of the phosphoramidate or phosphoramidite from cleaving orreacting during oligonucleotide synthesis. Those skilled in the art willrecognize that cyanoethyl moieties are preferred phosphate protectinggroups for their stability under oligonucleotide synthesis conditionsand their ease of removal with ammonia or methylamine. However, it willbe understood that because the phosphoramidate or phosphoramiditelinkage of the type utilized in the present invention need not bedeprotected and thus alkyl moieties generally or aryl containingmoieties are also suitable phosphate protecting groups R₄. It iscontemplated that vicinal groups —XR₁ and —YR₂ are most effective whenthey are positioned cis with respect to each other (R₁ and R₂ are H orsubstitution groups). Since adjacent functionalities attached to ringmoieties can be present in a cis configuration, and preferably a ringmoiety and —XR₁ and —YR₂ are oriented in space in a fixed cis position.However, —XR₁ and —YR₂ can be from straight chained moieties havingsuitable vicinal constituents, such as glycerol.

Those skilled in the art will appreciate that because of theiravailability on sugars and glycerol type diols, and because of knownprotecting groups suitable for their protection, X and Y are preferablyO (oxygen). However, it will be apparent to those skilled in the artthat utilizing NH and S in such positions for the purposes of thepresent invention is within the scope of the present invention.

In order to block one of the vicinal positions from participating in theoligonucleotide synthesis, R₁ or R₂ of vicinal OR₁ and OR₂ are suitableblocking groups. Because, as described below, the unblocked oxygen isactive in the final oligonucleotide cleaving step, the blocking groupshould be easily removed under cleaving reaction conditions but stableunder those conditions typically found in oligonucleotide synthesis. Forthis reason one of R₁ or R₂ is preferably an alkylcarbonyl orarylcarbonyl, such as acetyl or benzoyl. An alkylcarbonyl moiety is analiphatic group terminating in C═O, wherein the aliphatic componentcomprises one (i.e., Acetyl) to about 10 carbon atoms. By anarylcarbonyl group is meant a residue comprising at least onehomoaromatic or heteroaromatic ring and terminating in C═O (e.g.,C₆H₅CO). The protecting groups R₁ or R₂, which are not a blocking group,are suitably any protecting groups which are easily removed so that theprotected group is available as the site for the introduction of a firstnucleoside during the initiation of oligonucleotide synthesis. Forpurposes of the present invention, the 4,4′-dimethoxytrityl (DMT) groupis particularly preferred. Other suitable groups include, but are notlimited to, the following: 4,4′,4″-tris-(benzyloxy)trityl (TBTr);4,4′,4″-tris-(4,5-dichlorophthalimido)trityl (CPTr);4,4′,4″-tris(levulinyloxy)trityl (TLTr);3-(imidazolylmethyl)-4,4′-dimethoxytrityl (IDTr); pixyl(9-phenylxanthen-9-yl); 9-(p-methoxyphenyl)xanthen-9-yl (Mox);4-decyloxytrityl (C₁₀ Tr); 4-hexadecyloxytrityl (C₁₆ Tr);9-(4-octadecyloxyphenyl)xanthene-9-yl (C₁₈ Px);1,1-bis-(4-methoxyphenyl)-1′-pyrenyl methyl (BMPM);p-phenylazophenyloxycarbonyl (PAPoc); 9-fluorenylmethoxycarbonyl (Fmoc);2,4-dinitrophenylethoxycarb only (DNPEoc); 4-(methylthiomethoxy)butyryl(MTMB); 2-(methylthiomethoxymethyl)-benzoyl (MTMT);2-(isopropylthiomethoxymethyl)benzoyl (PTMT);2-(2,4-dinitrobenzenesulphenyloxymethyl)benzoyl (DNBSB); and levulinylgroups. These and other suitable protecting groups are described indetail in Beaucage, S. L. and Iyer, R. P. Tetrahedron 48, 2223-2311(1992), the entire disclosure of which is hereby incorporated byreference.

In particularly preferred embodiments of the present invention, theanchor monomer has the following structure:

Wherein R is H or CH₃. For purposes of the present invention, Brepresents a pyrimidine or purine base. Preferred for use in accordancewith the present invention are those bases characteristic of guanine,adenine, thymine and cytosine; however, other purine or pyrimidine basesas may be employed in the synthesis of nucleotide analogs mayalternatively be used as group B.IV. Polymer Synthesis

The solid substrate-linker-anchor moiety or solid substrate-linkercompounds described above serve as useful universal supports for thesynthesis of polymers (e.g., polynucleotides and polyamino acids). Ingeneral, the polymers may be synthesized by any means known in the art,including phosphoramidite mediated synthesis, photolithography (see,e.g., U.S. Pat. Nos. 5,424,186 and 5,744,305, each of which isincorporated herein by reference) or photogenerated acid mediatedsynthesis in combination with selective irradiation by a spatial opticalmodulator (See, e.g., WO 99/41007, incorporated herein by reference).Materials and protocols for phosphoramidite mediated synthesis ofoligonucleotides are well known in the art and available from GlenResearch, Sterling Va. Phosphite triester and H-phosphonate chemistriesare commonly used to prepare oligonucleotides on a solid support orsubstrate. Large scale commercial DNA synthesizers that employ phosphitetriester chemistry, have made the production of multi-kilo grams ofoligonucleotides possible.

Nucleosides used in large scale synthesis of oligonucleotides on a solidphase by phosphoramidite chemistry use are protected with suitablegroups that prevent formation of side products during oligonucleotidessynthesis. The reactive exocyclic amine groups found on the nucleobasesin monomer building blocks are generally protected with benzoyl,isobutyrl, phenoxyacetyl, and acetyl protecting groups, while thephosphate groups are usually protected as 2-cyanocthyl phosphoramidites.Such protective groups are easily removed after completion of theoligonucleotide synthesis by treatment with a concentrated solution ofammonium hydroxide.

The oligonucleotide is assembled by sequential addition of5′-dimethoxytritylated-3′-nuleooside phosphoramidites to the unmasked5′-hydroxy group of the first nucleoside loaded on to the support. Thisaddition is catalyzed by a mildly acidic catalyst such as tetrazole ordicyanoimidazole. The corresponding phosphite triester internucleotidelinkage is then converted to a more stable phosphate triester byoxidation with iodine or peroxides. “Capping” of any unreacted5′-hydroxyl groups by converting them to corresponding esters isachieved by a brief exposure to capping reagents containing aceticanhydride. Next, removal of 5′dimethoxytrityl group from the newly addednucleoside under mildly acidic conditions generates the 5′-hydroxylgroup and completes the coupling cycle. Using this method, a couplingefficiency of greater than 99% in each coupling step can be achieved.Towards the end of oligonucleotide synthesis, the dimethoxytrityl groupof the terminal nucleotide at the 5′-end is either left intact(“trityl-on”) or cleaved to give an oligonucleotide with free5′-terminal hydroxyl group (“trityl-off”). The 5′-trityl group may beused as a lipophilic purification handle to purify the full-lengtholigonucleotide bearing the trityl group from shorter and non-tritylatedspecies by reverse HPLC. After completion of oligonucleotide synthesis,the succinic ester linkage is cleaved under alkaline conditions torelease the oligonucleotide from the substrate in addition to theremoval of protective groups from the nucleobases and the phosphatebackbone. This process usually takes about 24 hours at room temperatureor about 6 hours at 55° C.

Several different methods for creating arrays of sequences on solidsupports (e.g., gene chips) are also known in the art. The universalsupports described above are useful as supports for array synthesis. Insome embodiments, the array synthesis is by photolithography methods(See, e.g., U.S. Pat. No. 5,143,854, incorporated herein by reference).In other embodiments, the array synthesis is performed by a masklessprocedure, such as those described in WO 99/41007 and WO 99/42813, eachof which is incorporated herein by reference. Each of these methodsemploy the light mediated deprotection or activation of reactive siteson the growing polymer chains in discrete, predefined regions.

The principles of solid phase chemical synthesis of polypeptides arewell known in the art and may be found in general texts in the area suchas Dugas, H. and Penney, C., Bioorganic Chemistry (1981)Springer-Verlag, New York, pgs. 54-92, Merrifield, J. M., Chem. Soc.,85:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis,pp. 24-66, Freeman (San Francisco, 1969). For example, polypeptides ofthe present invention may be synthesized by solid-phase methodologyutilizing an Applied Biosystems 430A peptide synthesizer (AppliedBiosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404)and synthesis cycles supplied by Applied Biosystems. Boc amino acids andother reagents are commercially available from Applied Biosystems andother chemical supply houses.

Accordingly, in some preferred embodiments, the present inventionprovides methods for synthesizing polymers, nucleic acids, peptides,carbohydrates, lipids, PNAs, on universal supports that comprise denselypacked linker groups that form a more ordered layer than previouslydescribed substrates. It is contemplated that the densely packed linkersare resistant to cleavage and increase the capacity of the solidsupport. Therefore, in some embodiments, the present invention providesmethods for synthesizing polymers comprising providing a substrate,stable linkers, an anchor group, and monomers, derivatizing thesubstrate with the stable linkers, attaching the anchor group to thelinkers to form a substrate-linker group-anchor group moiety, andsynthesizing a polymer from the monomers on the substrate-linkergroup-anchor group moiety. In preferred embodiments, the monomers areprotected monomers and synthesis proceeds deprotecting the protectedmonomers under conditions which do not cleave the polymer from thesubstrate, adding the desired protected monomer, and repeating until thedesired polymer is synthesized.

In still further preferred embodiments, the present invention providesmethods for controlling the number of polymers synthesized in adesignated area. In these embodiments, a co-coupling agent that is achain terminator that is similar in structure to the monomers is used inthe synthesis reaction. When included in an appropriate ration, theco-coupling reagent forms a number of inactive sequences within thegiven region that cannot be extended. Examples of co-coupling agentsuseful in the present invention include nucleophosphoramidites andnucleophosphonates that cannot be extended, for example 5′-MeO-T (SeeFIG. 2).

V. Polymer Deprotection, Washing, and Release

The present invention also provides improved methods for deprotecting,washing, and releasing polymers synthesized on the derivatizedsubstrate. In particular, as described above, the anchor moiety isselectively cleavable. As the growing polymer chain is continuallydeprotected under basic conditions, such as EDA in anhydrous EtOH, thepolymer attached to the substrate is not cleaved from the substrate.Upon completion of the deprotection reactions, the substrate surface isrinsed to remove small molecular fragments resulting from thedeprotection. This provides a surface with the polymer attached that isfree of salt and other small molecular contaminants. The polymers arethen removed from the substrate through a neighboring group assistedreaction, for example, 2-OH assisted 1-phosphate hydrolysis. Preferablythe cleavage agent is volatile (e.g., it can be removed via freezedrying) and non-ionic. The cleaved oligonucleotides are then recoveredby rinsing the substrate surface and the solution evaporated. When thepolymer is a oligonucleotide, the oligonucleotide are suitable for useas primers, templates, diagnostic probes, mass analysis and otherapplications, such as any enzymatic process, including DNA replication,reverse transcription, primer extension, phosphorylation, ligation,phosporylation, cleavage by restriction enzymes, etc., as naturallyoccurring oligonucleotide sequences. Previously described cleavage stepsrelied on the use of solvents containing metal ions such as Mg²⁺ orPb²⁺. The presence of these metal ions may deleterious to someprocedures such as mass analysis and enzymatic reactions. The washingsteps of the instant invention do not introduce appreciable amounts ofmetal ions.

Accordingly, in some embodiments, the present invention provides methodsfor cleaving and washing synthesized oligonucleotides comprisingproviding a solid substrate having attached thereto alinker-anchor-oligonucleotide moiety, washing the oligonucleotide on thesubstrate after synthesis and deprotection, cleaving the oligonucleotidefrom the linker-anchor-oligonucleotide moiety by a preferred reaction,and recovering the oligonucleotide, wherein the oligonucleotide ispreferably purified and more preferably substantially pure andsubstantially free of metal ions.

Accordingly, in some embodiments, the present invention provides methodsfor cleaving and washing synthesized oligonucleotides comprisingproviding a solid substrate having attached thereto alinker-anchor1-oligonucleotide1-anchor2-oligonucleotide2 moiety, washingthe sequence on the substrate after synthesis, cleaving oligonucleotide2by selectively cleaving the anchor2 moiety, recovering oligonucleotide2,cleaving oligonucleotide1 by selectively cleaving the anchor1,recovering oligonucleotide1, wherein the oligonucleotides aresubstantially pure and free of metal ions.

The present invention provides method of obtaining at least more thanone oligonucleotide from a single synthesis by incorporation of anchormoieties of different cleavage requirements into the sequence. The useof a variety of anchor moieties is contemplated, including, but notlimited to, those of the vicinal diol derivatives and dU in combination.dU can be first cleaved using UDG enzyme followed by amine treatment torelease one oligonucleotide. The second anchor, vicinal diol derivative,is deprotected (i.e., O-TBDMS or O-fpmp can be deprotect by extensiveacid treatment), rendering the diol moiety sensitive to basicconditions; the second oligonucleotide is released from substrate.Wherein the oligonucleotides are substantially pure and free of metalions.

VI. PCR Using the Oligonucleotides Synthesized

Accordingly, in some embodiments, the present invention provides methodsfor selective cleaving and recovering synthesized oligonucleotides in aform without contamination of the by products formed from deprotectionof nucleobase and phosphate protecting groups. In some embodiments ofthe present invention, the U anchor moiety has its 2′ or 3′ OH availablefor polymer synthesis. The cleavage after the synthesis, deprotectionand washing yields 3′-OH oligonucleotides and the 2′,3′-cyclophosphatebyproduct. One application of the 3′-OH oligonucleotides recovered afteron surface deprotection and washing are DNA polymerase substrates usedin PCR reactions to give desired DNA amplication (FIG. 11). The sequencegenerated using the stable diol linker without using any separatepurification produced the same PCR results as the primers obtained fromregular DNA synthesis.

VII. Ligation Using Oligonucleotides Synthesized

Accordingly, in some embodiments, the present invention provides methodsfor selective cleaving and recovering synthesized oligonucleotides in aform without contamination of the by products formed from deprotectionof nucleobase and phosphate protecting groups. These oligonucleotidescan find applications in making large DNA fragments or synthetic genes.According to the present invention, an assembly of oligonucleotides,whose sequences are derived from a gene and which can form staggeredpartial duplexes, are synthesized using a stable linker, such asliner-U-2′(3′)-OH (FIGS. 7 and 8). The syntheses, deprotection, washingand cleavage are performed as described, except for a 5′-phosphate isdirectly incorporated in the last step of synthesis using a compound ofthe structure:

For high efficient production, these syntheses may be carried on a DNAsynthesizer having parallel synthesis capability of 32 or more columnssimultaneously or on a microchip where each reaction site can beutilized to generate different sequences (Gao et al. (2001) NucleicAcids Res. 29, 4744-4750, cited herein entire reference). If ispreferable to use the linker of higher density, such as the C8 alkenyllinker, to maximize the sequences generated per unit surface area.

In one embodiment of the present invention,2′,3′-O-methoxyethylideneuridine or 2′,3′-O-methoxymethylideneuridine isprepared as described and converted to the 5′-phosphoramidite. The Ulinkage is formed by coupling the 5′-O-phosphoramidite U with thesurface OH group through the phosphate bond formation (FIG. 7; step 2).

A typical synthesis process is as follows:

Reaction Reagent/Solvent Special Steps Detritylation 3% TCA/CH₂Cl₂ orPGA- Use of PGA-P in parallel synthesis Wash CH₃CN, CH₃CN Activationtetrazole/CH₃CN Coupling monomer/activator/CH₃CN Special monomers, suchas 5′- phosphoramidite-U can be incorporated Wash CH₃CN Capping 10%acetic anhydride/THF (simultaneous) 10% Melm/THF/Pyridine(8/1) WashCH₃CN

PGA-P is a photogenerated acid precursor, such as triarylsulfoniumhexa(pentafluorophenyl)antimonite.

The synthesis of oligonucleotides is thus the same as conventionalsynthesis, but parallel synthesis on a microchip requires the in situformation of photogenerated acid (PGA) rather than as opposed to acid instandard DNA synthesis chemistry (Gao et al. (2001) Nucleic Acids Res.29, 4744-4750). The 2′,3′-ortho ester of U is then hydrolyzed upontreatment of 80% HOAc/H₂O at r.t. for 1.5-2.5 h to free one of thevicinal OH groups (FIG. 7, step 3) to provide an anchor point forpolymer growth.

The U-support prepared as described above, either on CPG in a column oron a microchip, is contacted with a 5′-DMT nucleophosphoramidite (A, C,G, or T, determined by the sequence synthesized) (FIG. 7, step 4). Thecoupling reaction results in formation of aU-2′(3′)-O-[Phosphite]-O-3′-N (N is the DNA monomer) linkage and thesequence is terminated with a 5′-DMT group. Following the capping, theoxidation, and the detritylation reactions, a second 5′-DMTnucleophosphoramidite monomer can be coupled to the 5′-OH on thesurface. The capping, oxidation, detritylation and coupling reactionsare repeated till the desired oligonucleotides are synthesized. Theoligonucleotide support is then treated with EDA/EtOH (1:1) to removebase and phosphate protecting groups as well as the 2′(3′)-acetyl group(FIG. 8, step 5). Tests have been performed using ³²P and T4 kinase tolabel the sequences potentially cleaved during the EDA treatment.However, electrophoresis analysis of the sample did not find any cleavedoligonucleotides. Thus, EDA does not cause hydrolysis of the2′(3′)-phosphate bond in U. After the deprotection reactions, theoligonucleotide surface is extensively washed with suitable solventsremove the small molecules formed from cleavage of the nucleobase andphosphate protecting groups (FIG. 8, step 6). Finally, theoligonucleotides are cleaved from the surface upon treatment withaqueous ammonium hydroxide, which hydrolyzes the 2′(3′)-cyclic phosphateto produce oligonucleotides with a free 3′-OH (FIG. 8, step 7). Thelinker-U moiety is also cleaved in this reaction, but they do not causeany problem in the subsequent enzymatic reactions. The reaction volumerecovered after cleavage reaction can be briefly evaporated to removeNH₃.

The oligonucleotides collected from either the solid support such as CPGor the microchips are directly used for ligation reactions without theneed de-salt purification. A set of oligonucleotides are mixed andannealed using temperature gradients, treated with enzymes such as Taqor T4 ligase for ligation, which joins the nicks in the long sequencescomprising of short, staggered partial duplex oligonucleotides alignedwith juxtaposed 3′-hydroxyl and 5′-phosphoryl end groups in anick-duplex structure. The optimal reaction condition for T4 DNA ligaseis 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 5%polyethyleneglycol-8000. In addition, T4 DNA ligase works adequately inthe presence of phosphorylation buffer. Therefore, it is not necessaryto remove the phosphorylation buffer if enzymatic phosphorylation isused. Taq DNA ligase can be used if the ligation needs to be done athigher temperature (˜65° C.).

The large synthetic DNA is separated from the short segments, which mayform due to non-specific hybridization, non-equivalent ligationefficiency, and other reasons. The large DNA duplex can be furtherpurified using match repair enzymes. The sequence accuracy will bevalidated using sequencing and agarose gel analysis. Further cloning andprotein expression are potential functional validation of the DNAsequence synthesized.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: amide linker: —(CH₂)₃NHCO(CH₂)₃X (X═OH, NH₂); ATP:adenosine triphosphate; Boc: ter-butyloxycarbonyl; C₃ linker —(CH₂)₃X(X═OH, NH₂); C₈ linker —(CH₂)₈X (X═OH, NH₂); CCD: charge coupled device;CPG: controlled porous glass; DCM: dichloromethane; DMF:dimethylformamide; DMT: 4,4′-dimethoxytrityl; DMT-Cl:4,4′-dimethoxytritylcholoride; EDA: ethylene diamine; Fmoc:9-fluorenylmethyloxycarbonyl; FR: fluorescence; FRE: fluorescenceemission; PGA: photogenerated acid; SSPE: (6′, 0.9 M NaCl, 0.066 MNaH2PO4, 0.012 M EDTA); TBE: (90 mM Tris-boric acid, pH 8.3, 2 mM EDTA;TCA: trichloroacetic acid; TEA: triethylamine; TEAA: triethylammoniumacetate; TFA: trifluoroacetic acid; Tris:tris(hydroxymethyl)aminomethane; THF: tetrahydrofunan; eq (equivalents);M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles);μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); C (degrees Centigrade); U (units), mU (milliunits); min.(minutes); sec. (seconds); % (percent); kb (kilobase); bp (base pair);PCR (polymerase chain reaction); BSA (bovine serum albumin), r.t. (roomtemperature).

EXAMPLE 1

This Example describes the derivatization of glass plates with an amidelinker. Microscope cover/slide glass plates and microarray platescontaining multiple sites were treated with hot piranha solution(concentrated H₂SO₄:H₂O₂, 50:50 v/v) for 15 min. The cleaned surface wasthoroughly rinsed with H₂O then EtOH, dried and immersed in a solutioncontaining N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (FIG. 1, amidelinker, 1% v/v in 95% EtOH). The reaction was left at room temperaturefor a minimum of 1 h with gentle shaking Upon completion of thereaction, glass plates containing the amide linker were rinsedthoroughly with 95% EtOH and cured at 100° C. under N₂ for 1 h. Thederivatized plates were stored in a clean, dry container.

EXAMPLE 2

This Example describes the derivatization of glass plates with anexemplary alkenyl linker (C₈). Microscope cover/slide glass plates andmicroarray plates containing multiple sites were treated with hotpiranha solution (concentrated H₂SO₄:H₂O₂, 50:50 v/v) for 15 min. Thecleaned surface was thoroughly rinsed with H₂O, acetone, CH₂Cl₂, thencyclohexane, and dried. The plates were placed in a sealable containercontaining 5 mM 7-octenyltrimethoxysilane, (FIG. 2,CH₂═CH(CH₂)₆Si(OCH₃)₃) in cyclohexane or 5 mM docosenyltriethoxysilane(FIG. 2, CH₂═CH(CH₂)₂₀Si(OCH₂CH₃)₃) in 4:1 cyclohexane:CHCl₃. Thereaction was left at room temperature with gentle shaking for 16 h. Uponcompletion of the reaction, glass plates were rinsed thoroughly withcyclohexane and cured at 100° C. under N₂ for 1 h. The derivatized glassplates were placed in a 10 mL glass vial closed by a septum, separatedby Teflon sheets. The container was purged with N₂ for 10 min, and 5 mLof 1 M BH₃.THF was introduced using N₂ and canulas. The borane reactionwas allowed for 2 h at r.t. under gentle shaking The solution wasremoved using N₂ positive pressure, and 5 mL of an oxidation solutionwas introduced. Three oxidation solutions were utilized: 0.1 M NaOH in30% H₂O₂ for 3 min at room temperature (Netzer, L., Iscovivi, R., Sagiv,J. Adsorbed monolayers versus Langmuir-Blodgett monolayers—why and how?I. From monolayer to multilayer, by adsorption. J. Thin Solid Films(1983) 99, 235-241; Netzer, L., Iscovivi, R., Sagiv, J. Adsorbedmonolayers versus Langmuir-Blodgett monolayers—why and how? II.Characterization of built-up films constructed by stepwise adsorption ofindividual monolayers. J. Thin Solid Films (1983) 99, 67-76; Netzer, L.,Sagiv, J. A new approach to construction of artificial monolayerassemblies. J. Am. Chem. Soc. (1983) 105, 674-6; Wasserman, S. R., Tao,Y.-T., and Whitesides, G. M. Structure and reactivity of alkylsiloxanemonolayers formed by reaction of alkytrichlorosilanes on siliconsubstrates. Langmuir (1989) 5, 1074-1087); 3 M NaOAc in 30% H₂O₂, pH 7.5for 10 hours at room temperature; and 1:10 30% H₂O₂:THF at 0° C. for 24h. The reaction solution was then removed and the plates were rinsedwith H₂O, EtOH, dried and stored in a clean, dry container

EXAMPLE 3

This Example describes the derivatization of glass plates. Microscopecover/slide glass plates and microarray plates containing multiple siteswere treated with hot piranha solution (concentrated H₂SO₄:H₂O₂, 50:50v/v) for 30 min. The cleaned surface was thoroughly rinsed with EtOH,CH₂Cl, and toluene, and dried with a stream of ultra high purity N₂. Theplates were placed in a closed container containing 43 mM3-aminopropyltriethoxysilane (FIG. 2) in toluene. The reaction washeated to 60° C. for 4 min. Upon completion of the reaction, glassplates were rinsed five time with toluene and dried with N₂.

EXAMPLE 4

This Example describes the analysis of contact angles of derivatizedglass plates. Contact angles were measured at r.t. by application ofstatic drops (4-10 μL) of deionized water to linker derivatizedsubstrate surfaces with a micropipetter. The measurements were madevisually on both sides of the drops using a Zisman type goniometerequipped with a video camera. Tangent to the drop at its intersectionwith the surface determined contact angle θ. The advancing contactangle, θ_(a), was taken as the maximum contact angle observed as thedrop size was incrementally increased without an increase in the contactarea. The receding contact angle, θ_(r), was taken as the minimumcontact angle observed as the drop size was decreased with a decrease inthe contact area. Average values of a least three measurements performedon each substrate were reported. These measurements are shown in Table1.

TABLE 1 Contact Angle of Monolayer Linker on Glass Substrate Linker(Terminus group) Advancing (°) Receding (°) Amide Linker (OH) 54 44 C₈(CH₂═CH—) 92 83 C₈ (OH)^(a) 63 53 C₈ (OH)^(a) 69 55 C₈ (OH)^(a) 66 55C₂₂ (CH₂═CH—) 100 74 C₂₂ BH₃/NaOH (OH)^(b) 92 58 C₃ (NH₂) 54 30C(CH₃)₂C₂ (NH₂) 55 52 C(CH(CH₃)₂)₂C₂ (NH₂) 65 54 ^(a)Hydroxyl group wasintroduced by oxidizing the terminus double bond using BH₃/THF/H₂O₂,BH₃/NaOAc, or BH₃/NaOH, correspondingly. ^(b)Hydroxyl group wasintroduced by oxidizing the terminus double bond using BH₃/NaOH.

EXAMPLE 5

This Example describes oligonucleotide synthesis on glass platesderivatized with the C₈ or amide linkers. The amide or C₈ linkerderivatized glass plates were divided into strips of ˜30 mm² andsynthesis was performed in a circular column. The glass plates were heldin the direction of flow by two pieces of Teflon inserts in the column.The surface of the inserts was caved to allow contact of reactionsolution with the surface of glass plates. The oligonucleotidessynthesis used an automated DNA synthesizer (Expedite 8909) andprotocols that were modified from that of standard 1 μmol synthesis. Atypical protocol for such a synthesis is given below (Table 2). TheDMT-monitor on the synthesizer was turned off because the amount of DMT⁺was too little to be measured. Monomers were DMT-dA(N6bz), DMT-dC(N4bz),DMT-dG(N2ib), and DMT-T.

TABLE 2 Synthesis Protocol of DNA Oligonucleotides on Glass Plates ConcReaction Reagent (mM) # Pulse Vol.(ml) Time Set Time(sec) Detritylation3% TCA/CH₂Cl₂  29.5 110 1.760 39.6 wash A CH₃CN na 170 2.720 37.4 WashCH₃CN anhydrous na 80 1.280 28.8 Coupling Activator: tetraazole/CH₃CN 450.0 35 0.560 12.6 Mono + activator activator  450.0 50 0.800 18.0(simultaneous) monomer + activator  50.0 50 0.800 Wash CH₃CN anhydrousna 8 0.128 2.9 wash A CH₃CN na 130 2.080 28.6 OxidationI₂/THF/pyridine/H₂O  20.0 60 0.960 21.6 Oxidation I₂/THF/pyridine/H₂O 20.0 10 0.160 15.000 15.0 wash A CH₃CN na 120 1.920 26.4 Capping 10%acectic anhydride/THF 1057.8 50 0.800 18.0 (simultaneous) 10%Melm/THF/Pyridine(8/1) 1254.6 50 0.800 Capping 10% acectic anhydride/THF1057.8 10 0.160 15.000 15.0 (simultaneous) 10% Melm/THF/Pyridine(8/1)1254.6 10 0.160 wash A CH₃CN na 140 2.240 30.8

EXAMPLE 6

This Example describes oligonucleotide synthesis on glass platesderivatized with the C₈ or amide linkers. Oligonucleotide synthesis isperformed with DMT-tri(hexaethylene glycol) phosphoramidite,DMT-dA(Nbz), DMT-dC(Nbz), DMT-dG(Nib), and DMT-T phosphoramidite, and5′-MeO-T phosphoramidite as the co-coupling agent of DMT-Tphosphoramidite.

DMT(O(CH₂)₂)₃OP(OCH₂CH₂CN)(NCH(CH₃)₂)₂ (spacer, DMT-hexaethyleneglycosylphosphoramidite) (FIG. 1) was prepared using the same reactionconditions as tritylation and phosphitylation of DNA nucleoside.5′-MeO-T (Kowollik, G., Gaertner, K., and Langen, P. (1966)5′-O-methylthymidine. Angew. Che. Internat. Edit. 5, 735-736) and5′-CH₃-T (Sekine, M., and Nakanishi, T. (1990) Facile synthesis of3′-O-methylthymidine and 3′-dexoythymidine and related deoxygeneratethymidine derivative: A new method for selective deoxygenation ofsecondary hydroxy groups. J. Org. Chem. 55, 924-928) (both are chainterminators) (FIG. 2) were prepared according the procedures describedin the literature. The corresponding phosphoramidites were prepared in asimilar manner as T phosphoramidite preparation.

The amide or C₈ linker derivatized glass plates were divided into stripsof ˜30 mm² and synthesis was performed in a circular column. The glassplates were held in the direction of flow by two pieces of Tefloninserts in the column. The surface of the inserts was caved to allowcontact of reaction solution with the surface of glass plates. Theoligonucleotides synthesis used an automated DNA synthesizer (Expedite8909) and protocols that were slightly modified from that of standard 1μmol synthesis (Table 2). The DMT-monitor on the synthesizer was turnedoff because the amount of DMT⁻ was too little to be measured. In somesynthesis, the first few steps of coupling were performed withoutcapping of the failure sequences. The synthesis steps used a mixture ofcoupling and co-coupling agents, such as T and 5′-MeO-T phosphoramiditesare indicated by X, where the coupling and co-coupling agents (e.g. aterminator) are in various ratios; ratio typical was 1:1-1:10. Examplesof the sequences synthesized on glass plates were given below (S=spacer;3′-tail=5′-TTTTT, 5′-XTTTTT, 5′-TTTXTT, 5′-SSTTTTT, 5′-XSSTTTTT,5′-SXSTTTTT, or 5′-SSXTTTTT).

15-mer: (SEQ ID NO: 1) 5′-TATGTAGCCTCGGTC-3′-tail 16-mer: (SEQ ID NO: 2)5′-CTCCTACGGGAGGCAG-3′-tail 24-mer: (SEQ ID NO: 3)5′-GTCACCATGTTGACTCACCATGTC-3′-tail 41-mer: (SEQ ID NO: 4)5′-TGTTGACTCACCATGTCGTCACCATGTTGACTC ACCATGTC-3′-tail

EXAMPLE 7

This Example describes the time dependent ammonolysis ofoligonucleotides from the solid support. The glass plates (ca. 2 mm²)containing T₁₀ in eppendorff tube were treated with conc. NH₄OH (50 μL)at r.t. At 15 and 30 min, the solution was removed from the tube. Theglass plates were treated with NH₄OH (50 μL) again for 16 h at r.t.These solution samples were vacuum dried and redissolved in H₂O (10 μLfor each 1 mm² plate). A portion of the sample (3 μL) was labeled withγ-³²P-ATP (5 μCi, 3000 Ci/mmole) using T4 polynucleotide kinase (1 u)and the conditions recommended by the manufacturer (Gibco). ³²P-labeledoligonucleotides (4 μL) were mixed with formamide (6 μL) before loadingonto a gel containing 20% acrylamide/bisacrylamide (29/1) in 7 M urea.Gels in 1×TBE (90 mM Tris-Boric acid, pH 8.3, 2 mM EDTA) were subjectedto electrophoresis at 55 V/cm for ˜1.5 h at room temperature. ³²Pexposure on an X-ray film (Kodak) produced gel films. The intensities ofthe gel bands were derived using the Image Pro program (Media Imagenics)after scanning digitization of the gel film. FIG. 3 displayselectrophoresis gel profiles of T₁₀ cleaved from glass plates at 15, 30and 60 min upon treatment with conc. aq. NH₄OH. The T₁₀ with amidelinker is shown on the left panel and the T₁₀ with C₈ linker is shown onthe right panel.

EXAMPLE 8

This Example describes an assay of oligonucleotide synthesis using atermination nucleophosphoramidite, 5′-MeO-T, to probe the presence ofavailable sites for coupling with a phosphoramidite at differentreaction stages. The sequences terminated with 5′-MeO-T are notobserved, since they cannot be ³²P labeled at the 5′-OH using T4polynucleotide kinase. The results of these assays are illustrated inFIG. 4. (A) Regular T₃ synthesis on glass plates. (B) Illustration ofthe use of termination monomer. T on glass plate is coupled with MeO-T,resulting in the formation of a terminated dimer T-T(OMe), which can notundergo further chain growth. (C) Illustration of the hypothesis forreaction with more hindered surface sites in several continued reactioncycles. If these sites exist, oligonucleotides can be synthesized evenafter applying MeO-T in the coupling step. (D) ³²P-gel electrophoresisanalysis of the experiments using the termination 5′-MeO-T at differentstages of oligonucleotide synthesis. Lane 1. Sequences from a synthesiswhich used MeO-T in the first step of coupling, followed by couplingwith DMT-T. The sites that failed to couple with MeO-T would produceregular sequences, such as T₃. This sequence is clearly present in asignificant ratio along with T₂ and T₁ fragments. Lane 2. Sequences froma synthesis which used MeO-T at the second step of coupling, followed bycoupling with DMT-T. The monomer T sites that failed to couple withMeO-T would produce regular sequences, such as T-T₃ or T₄. In thisexperiment, little T₄ was observed. The surface OH sites that failed tocouple with DMT-T in the first step would also be responsible for theobserved T₁₋₃ sequences. Lane 3. Sequences from a synthesis which usedMeO-T at the third step, followed by coupling with DMT-T. T₅ and T₄ werenot observed. There are diminished amounts of overall sequences andshort T_(n) fragments. Lane 4. Sequences from a synthesis which usedMeO-T at the fourth step, followed by coupling with DMT-T. Only minorT₁₋₃ were observed. Lane 6. Regular synthesis of T₆ as a control.

EXAMPLE 9

This Example describes hybridization of complementary sequences to thesynthesized sequences. The target sequences (100-200 nM) containingfluorescein label were dissolved in a 6× SSPE solution (50-200 μL, 1 MNaCl, 66 mM sodium phosphate, 6 mM EDTA, pH 7.4) and applied to theglass plate or a chip containing probe sequences. The experiments wereperformed under a cover slip at r.t. or a temperature suitable for thegiven set of target and probe sequences for 2 h or longer. The plateswere then washed twice with 6× SSPE, spin dried, and the fluorescenceimage was taken using a cooled CCD camera (Apogee Instruments). A 200 WXenon lamp was used as the light source. Fluorophore excitation anddetection were 475 and 535 nm, respectively. Fluorescence images wereprocessed and analyzed using the Image Pro (Media Cybernetics),ScanAlyze2 (http://rana.Stanford.EDU/software/), and the Excel(Microsoft) programs. Fluorescence intensities were reported afterbaseline correction and averaging over redundant data points.

EXAMPLE 10

This Example describes hybridization of complementary sequences to thesynthesized sequences. The glass pates containing the 24-mer and the41-mer probes were hybridized with target sequences as described andwere then washed with low salt buffer solution containing NaCl (5 mM)and NaH₂PO₄ (5 mM), pH 7.0 until the fluorescence intensity reading wascomparable to background of the glass plates. The hybridization andimage acquisition were repeated multiple times. The comparison of theprobe sequences synthesized using the amide and C8 linkers and used forthree time hybridization experiments is shown in FIG. 5.

EXAMPLE 11

This Example describes synthesis on CPG using the C₈ and amide linkers.

(a) CPG-O₃Si(CH₂)₈OH (C₈ linker)

CPG (500 Å, 500 mg or 2000 Å, 500 mg) in 2.5%7-octenyltrimethoxysilane/cyclohexane was shaken at rt for 24 h, thenwashed with cyclohexane, dried at 100° C. for 0.5 h in vacuo. Thederivatized CPG (100 mg) was treated with borane/THF (1.0 M, 2.5 mL) atrt under N₂ with occasional shake for 3 h. CPG was washed with THF.Unreacted borane/THF was destroyed with ice H₂O. CPG was then treatedwith 0.1 M NaOH/30% H₂O₂ (1:1) at rt for 3 min. The solution was removedby filtration. CPG was washed with H₂O, EtOH, and acetone, and driedunder vacuum.

(b) CPG-O₃Si(CH₂)₃NHCO(CH₂)₃OH (amide linker)

CPG (500 Å, 500 mg) in 2% (EtO)₃Si(CH₂)₃NHCO(CH₂)₃OH/95% EtOH was shakeat rt for ˜12 h, washed with 95% EtOH and diethyl ether, and cured onhot plate (˜100° C.) for 1 h with N₂.

CPG loading: The linker derivatized CPG (10 mg) in dry pyridine (0.5 mL)was shaken with DMTCl (10 mg) at rt for 3 h and then washed, insequence, with pyridine, sat. NaHCO₃ in ice H₂O (1:1), H₂O, EtOH,CH₂Cl₂. The tritylated CGP was then treated with 2% TCA for 2 min and aportion of the resultant DMT⁺/CH₂Cl₂ solution was measured at 503 nm(ε=76 mM cm⁻¹). Calculation was performed to obtain the loading of CPG(μmol linker-OH sites/g). The results were 20 μmol/g for CPG-amidelinker (500 Å), 108 μmol/g for CPG-C₈ linker (500 Å), and 22 μmol/g forCPG-C₈ linker (2000 Å).

EXAMPLE 12

This Example describes synthesis on CPG using C₈ and amide linkers,wherein the first coupling is with a uridine moiety.2′,3′-O-methoxyethylideneuridine or 2′,3′-O-methoxymethylideneuridinewas prepared according to literature (Fromageot, H. P. M., Griffin, B.E., Reese, C. B., Sulston, J. E. The synthesis ofoligoribonucleotides-III. Monoacylation of ribonucleosides andderivatives via orthoester exchange. Tetrahedron 1967, 23, 2315-2331)(FIG. 6). These compounds were converted to 5′-phosphoramidites using asimilar procedure to that for preparation of DNA nucleophoramidites(FIG. 2). The 5′-U phosphoramidite was freshly dissolved in CH₃CN (50mM) and placed on a DNA synthesizer for automated synthesis ofoligonucleotides.

Oligonucleotide synthesis used derivatized CPG (Table 3) containingstable amide or C₈ linker and was performed in a 1 μmol column. 5′-Uphosphoramidite was coupled to the linker terminus OH group using theRNA synthesis protocol including coupling, capping and oxidation steps(FIG. 7). The 2′,3′-ortho ester of U was hydrolyzed after treatment of80% HOAc/H2O (1 mL) at r.t. for 1.5-2.5 h (FIG. 7). The CPG was washedwith cold H₂O, saturated ice NaHCO₃/H₂O (1:1), cold H₂O, and CH₃CN,dried in vacuo. The linker-5′-U derivatized CPG (Table 3) was loadedinto a 1 μmol column on a DNA synthesizer. Oligonucleotide sequenceswere synthesized using standard synthesis protocols.

Upon completion of synthesis, the sequence bound CPG was treated withEDA/EtOH (1:1, v/v, 1 mL) at r.t. for 2 h, washed with 95% EtOH andCH₃CN, and dried in vacuo to give protecting group free sequence boundCPG (FIG. 8).

The deprotected or protected sequence bound CPG was treated with one ofthe following conditions: (i) NH₄OH (aq. 0.5 mL) at 80° C. for 8 h; (ii)or conc. NH₄OH/40% MeNH₂ (1:1) at 80° C. for 3 h; (iii) 40%MeNH₂/TEA/EtOH (1:1:0.2) at 80° C. for 3 h; (iv) conc. NH₄OH/TEA/EtOH(1:1:0.6) at 80° C. for 3 h. The solution containing cleavedoligonucleotides (3′-OH) was dried by speed-vac. The residue wasdissolved in H₂O and centrifuged. The aqueous solution was analyzed byHPLC. The analysis result showed that conc. NH₄OH at 80° C. for 8 h is abetter condition for cleaner cleavage and that C8 linker produced threetimes more oligonucleotides than that produced using the amide linker onthe same amount of CPG (Table 3). The primer KB12 sequences synthesizedusing the stable amide linkers or the regular succinyl linker gave thesame HPLC retention times.

HPLC was performed on a C₁₈ reverse phase column (Waters) using 50 mMTEAA in CH₃CN. Flow rate was 1 mL/min. HPLC results are shown in FIG. 9.Mass data were obtained from a MALDI-TOF instrument (Profelix, Bruker)in negative mode and these results are given in Table 3.

TABLE 3 Summary of Oligonucleotide Synthesis on CPG Using Non-cleavable Linkers Coupled to 5′-Phosphoramidite-U Cleavage Amount MS Sequence On Support from Cleavage Product Appli- (calc.; No CPG Linker(5′-3′)^(a) Deprotection Support Detritylation from U (OD) HPLC cationobs.) 1 500 A, C8-rU 5′-DMT- EDA/EtOH ammonia 80%   ammonia 10 mg,primer (1:1)  (aq),  AcOH, (aq),  1 umol KB12,  rt, 2 h rt, rt., 80 C.,38 20-mer 48 h 1 h 8 h 2 2000 A, C8-rU 5′-DMT- EDA/EtOH ammonia 80% ammonia 23 fig PCR 6084; 23 mg, primer (1:1)  (aq),  AcOH, (aq),  60650.5 umol KB12, rt, 2 h rt, rt., 80 C., 20-mer 48 h 1 h 8 h 3 500A, C8-rU5′-DMT- EDA/EtOH ammonia 80%  ammonia 34 10 mg, primer (1:1)  (aq), AcOH, (aq),  1 umol KB12, rt, 2 h rt, rt., 80 C., 20-mer 48 h 1 h 8 h 42000 A, C8-rU 5′-OH half  75 fig 18591; 23 mg, HIVs60mer of #4, 185320.5 umol ammonia (aq),  80 C., 8 h 4a from #4, C8-rU 5′-FR EDA/EtOHammonia fig hybridi- 0.25 HIVs60mer (1:1)  (aq),  zation umol rt, 2 h80 C., 8 h 4b from #4, C8-rU  5'-FR EDA/EtOH enzyme fig hybridi- 0.25HIVs60mer (1:1)  purifi- zation umol rt, 2 h cation, then ammonia (aq), 80 C., 8 h 6 500 A, amide 5'-OH- EDA/EtOH ammonia 28 25 mg, primer(1:1)  (aq),  0.5 umol KB12, rt, 2 h 80 C., 20-mer 8 h 6a from #6,ammonia 22 500 A, amide 5'-OH- (aq),   25 mg, primer 80 C., 0.5 umolKB12, 8 h 20-mer ^(a)primer KB12: 5′-TCTATTGTTGGATCATATTC (SEQ ID NO:5); HIVs60mer5′-TGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCT (SEQ IDNO: 6)

EXAMPLE 13

This Example describes purification of the synthesized oligonucleotideswith enzymes. The synthesis of HIVs6Omer, 5′-FR-TGG AAA GAT ACC TAA AGGATC AAC AGC TCC TGG GGA TTT GGG GTT GCT CTG GAA AAC TCT (SEQ ID NO: 7)(FR=diisobutyryl-4(5)-CO-fluorescein-NH(CH₂)₆OP(O₂)—), was performed asdescribed above (Table 3) except that the final coupling usedfluorescein phosphoramidite. The FR-HIVs60mer bound CPG was directlytreated with conc. NH₄OH at 80° C. for 8 h to give free 5′-FR-HIVs60mer.Another portion of FR-HIVs60mer bound CPG (2 mg) was deprotected usingEDA/EtOH, washed with EtOH, and then treated with phosphodiesterase IIaccording to the procedures described (Gao, X., Zhang, H., and Zhou, X.Method of oligonucleotide purification using enzymes. U.S. patentapplication Ser. No. 09/364,643). The enzyme purified sequence wascleaved from CPG using NH₄OH at 80° C. for 8 h. HPLC (FIG. 10) wasperformed on a C₁₈ reverse phase column (Waters) using 50 mM TEAA inCH₃CN. Flow rate was 1 mL/min.

EXAMPLE 14

This Example describes PCR using the sequences synthesized above.Aliquots of 50 μL of PCR reaction mixture contained 1 pg template DNAstrand (1 μL, 30 pM, Ras 99-mer), 11 pmol of each corresponding primer(KB12, 20-mer made on CPG with either stable amide-U linker or regularcleavable succinyl linker), 10 mM of each dNTP (4 μL), and 2 μL Taqenzyme (Promega) in storage buffer. Amplification was carried out for 30cycles of 90° C. for 30 s, 53° C. for 30 s, and 72° C. for 30 s. Uponcompletion of the reaction, DNA was purified from the unincorporateddNTPs and the primer with a Clontech purification kit (chromaspin+TE-30) according to the manufacturer's protocol.

The analysis of the PCR products from the two primers (FIG. 11) used 2%agarose gel and TBA buffer. The sequence generated using the stable diollinker without using any separate purification produced the same PCRresults as the primers obtained from regular DNA synthesis.

EXAMPLE 15

This Example describes the derivatization of various surfaces withlinking groups.

General Methods

Contact angle measurements. Contact angles were measured by applicationof static drops (4-10 μL) of deionized water to the substrate surfaceswith a micropipetter. The measurements were made visually on both sidesof the drops using a Zisman type goniometer. The advancing contactangle, θ_(a), was taken as the maximum contact angle observed as thedrop size was incrementally increased without an increase in the contactarea. The receding contact angle, θ_(r), was taken as the minimumcontact angle observed as the drop size was decreased with a decrease inthe contact area. The average values of a least three measurementsperformed on each substrate were recorded.

The contact angle measurements were performed using the SCA20 software(DataPhysics Instruments GmbH). Droplet images were acquired with a CCDvideo camera module (SONY, model XC-77CE). Droplets were dispensed witha Multielectrapette pipette (Matrix technologies).

Imaging of glass plates. After labeling with 4(5)-carboxyfluoresceindiisobutyrate, glass plates were treated with ethylene diamine (50% inabsolute EtOH) for 15 min, followed by washing with EtOH and acetone anddrying using dry N₂. The plates were placed on a microscope slide undera cooled CCD camera (Apokee Instruments). The fluorophore was excitedand detected at 494 and 525 nm, respectively. The light source was a 200W Hg—Xe lamp (model 66033, Oriel Instruments). Light exposure time wasfrom 10 to 60 sec. The fluorescent images of the plates were acquired,processed and analyzed using the Image Pro program (Media Imagenics).

Glass derivatization (functionalization of the substrate). Glass plates(22×22 mm²) were cleaned using piranha solution (H₂SO₄/H₂O₂, 1:1) for 30min. After rinsing thoroughly with 18 mΩ water, the plates are carefullyrinsed with EtOH 95%, DCM, toluene and dried with a stream of ultra highpurity N₂.

Glass plates were dipped in a solution of 3-aminopropyltriethoxysilane(43 mM) in toluene heated to 60° C. for 4 min. Following the reaction,glass plates were washed 5 times with toluene and dried with N₂.

Fluorescence labeling and quenching issues. Amino groups attached toglass plate surface can be labeled with 4(5)-carboxyfluoresceindiisobutyrate and detected by fluorescence emission. However, closeproximity of the fluorescent molecules on the flat surface can result influorescence quenching. Thus, optimal conditions for fluorescentlabeling had to be determined. A glass plate (22×22 mm²) derivatizedwith 3-aminopropyltriethoxysilane (procedure 1 and 2) was cut in piecesof 3×2 mm². Each of the pieces were placed in 0.6 mL propylene testtube. Fluorescent labeling was carried out with 100 μL of4(5)-carboxyfluorescein diisobutyrate with Boc-Gly-OH (a total of 6 μmolfor the two species), HOBt (0.91 mg, 6 μmol, prepared from aconcentrated solution), and DIC (1 μL, 6 μmol, prepared from aconcentrated solution). Boc-Gly-OH was used to compete with4(5)-carboxyfluorescein diisobutyrate for the coupling with the freeamino groups present on the flat substrate. Thus, by varying the ratioof the two reagents (keeping the total concentration of active speciesconstant), the concentration of 4(5)-carboxyfluorescein diisobutyrate onthe surface can be diluted. Reactions were carried out for 5 and 60 min(FIG. 1).

High concentration of 4(5)-carboxyfluorescein diisobutyrate inducesfluorescence quenching. In the case of glass plates obtained fromprocedure 1, the mean fluorescence intensity varies linearly with4(5)-carboxyfluorescein diisobutyrate concentration for concentration ashigh as 48 mM (80% of 60 mM) for a 5 min reaction time. However, themean fluorescence intensity drops for the 60 mM 4(5)-carboxyfluoresceindiisobutyrate sample. This effect is more critical as the reaction timeincreases to 60 min. In this case, linearity is conserve for only up to18 mM (30% of 60 mM) 4(5)-carboxyfluorescein diisobutyrate. Thissuggests that 4(5)-carboxyfluorescein diisobutyrate density on thesurface increases as reaction time increases.

The results appear to be dependent on the glass plate derivatizationprocedure and on the initial density of 3-aminopropylsilane. When theexperiment was reproduced on a glass plate derivatized with3-aminopropyltriethoxysilane according to procedure 2, linearity isconserved only for concentration of 4(5)-carboxyfluoresceindiisobutyrate below 9 mM. Above this concentration, fluorescencequenching occurs and fluorescence intensity drops. In this case, thiseffect seems to be independent of the reaction time. Therefore, thissuggests that 4(5)-carboxyfluorescein diisobutyrate and Boc-Gly-OHcoupling to the surface is achieved in high yield within the first fiveminutes and that increasing the reaction time has little effect on thefluorescent moiety surface density.

Although 3-aminopropyltriethoxysilane is common to the twoderivatization procedures, it seems that two different types of surfacesare obtained: one with low reactivity (from procedure 1) and one withhigh reactivity (from procedure 2). Thus, procedure 2 is suitable forpeptide synthesis on glass surface. Furthermore, 4(5)-carboxyfluoresceindiisobutyrate (9 mM) diluted with Boc-Gly-OH (51 mM) and reacted for 5min with the glass plate are optimal conditions for the fluorescentlabeling of free amino groups present on the glass plate. It is assumedhere that the free amino group surface density is maximum afterderivatization and that subsequent peptide synthesis would lead to adensity equal or lower to this starting density. However, other factorsthan density may be involved in the quenching process. It appears forinstance that fluorescence emission increases as the distances betweenthe fluorescent moiety and the surface increases.

EXAMPLE 16

This Example describes the stability of silane bond to chemicalsinvolved in peptide chemistry. Prior to peptide synthesis on the flatsolid support, stability of the Si—O—Si bonds towards acidic and basicchemicals involved in peptide chemistry needed to be assessed. A glassplate derivatized from procedure 2 was cut into 3×2 mm² pieces. Each ofthe pieces were placed in 0.6 mL propylene test tubes and reacted with avariety of reagents. Following reaction, glass plates were fluorescentlylabeled according to the conditions described earlier and fluorescentemission was recorded. Reagents tested are summarized in Table 4.:

Density control—coupling with a dentrimer can increase the density

TABLE 4 Glass Reaction Mean Fluorescence Plate # Reagents time Intensity(arbitrary unit) 1 TMSOTf (19.5 μL, 107  16 h. 6000 ± 200 μmol) TFA (69μL, 895 μmol) m-cresol (12 μmol, 115 μmol) 2 Piperidine (20 μL, 200 16 h5900 ± 200 μmol) DMF (80 μL) 3 TEA (10 μL, 71 μmol) 16 h 6000 ± 200 DCM(90 μL) 4 No reagents, for control 6100 ± 200 5 No reagents, for 6000 ±200 reproducibility

According to the mean fluorescence intensity measured for each samples,the density of aminopropylsilane on the glass surface is constant. Theconditions tested do not seem to induce cleavage of the linker. Peptidesynthesis can be carried out without damaging the surface.

EXAMPLE 17

This Example describes glass derivatization with11-bromoundecyltrimethoxysilane. Glass plates (22×22 mm²) were cleanedusing piranha solution (H₂SO₄/H₂O₂, 1:1) for 30 min. After rinsingthoroughly with 18 mΩ water, the plates are carefully rinsed with 95%EtOH, DCM, cyclohexane and dried with a stream of ultra high purity N₂.

The glass plates were dipped in a solution of11-bromoundecyltrimethoxysilane (63.5 μL, 2 mM) in cyclohexane (100 mL)at room temperature for 5, 60, and 270 minutes. Following reaction,glass plates were washed 2 times with cyclohexane, washed with hotcyclohexane (80° C.) for 5 min, rinsed with DCM, acetone, EtOH, anddried with N₂.

EXAMPLE 18

This Example describes in situ modifications.

Azide-terminated linker on solid support. Glass plates containingBromide terminated C₁₁ linker as described above were placed in asupersaturated solution of NaN₃ in dry DMF (1.5 g in 100 mL). Thesolution (together with the undissolved NaN₃) was stirred at roomtemperature. After 24 h the glass plates were rinsed with distilledwater.

Amino-terminated linker on solid support. The above azide-terminatedglass plates were placed in lithium aluminum hydride solution (0.2 M inTHF). After 24 h the glass plates were soaked in THF for an additional24 h. The glass plates were placed in 5% HCl solution for 5 h tocomplete hydrolysis of the aluminum complexes, rinsed with deionizedwater, acetone, and placed in TEA for 10 min in order to convert theterminal —NH₃ ⁺ into —NH₂.

EXAMPLE 19

This Example describes cleavage of the linkers from the glass plates.Glass plates samples (3×2 mm²) were treated with NH₄OH (29%, 50 μL) atroom temperature, washed with water, 1% TFA in DCM, 10% TEA in DCM, andlabeled with diisobutyrate carboxyfluorescein. The experimental contactangles are compared to those described in Heise, A.; Menzel, H.; Yim,H.; Foster, M.; Wieringa, R. H.; Schouten, A. J.; Erb, V; Stamm, M.Grafting of Polypeptides on Solid Substrates by Initiation ofN-Carboxyanhydride Polymerization by Amino-Terminated Self-AssembledMonolayers. Langmuir 1997, 13, 723-728; and Fryxell, G. E.; Rieke, P.C.; Wood, L. L.; Engelhard, M. H.; Williford, R. E.; Graff, G. L.;Campbell, A. A.; Wiacek, R. J.; Lee, L.; Halverson, A. NucleophilicDisplacements in Mixed Self-Assembled Monolayers. Langmuir 1996, 12,5064-5075.

TABLE 5 Contact Angle Measurements Glass Experimental Reported PlatesAdvancing Receding Advancing Receding Br 5′ 76 72 82 77 Br 60′ 75 71 Br270′ 81 70 N3 Br 5′ 75 69 77 71 N3 Br 270′ 78 72 NH2 Br 5′ 72 45 63 42NH2 Br 270′ 73 44 NH2 silane 1 70 40 NH2 silane 2 61 32 NH2 silane 3 5428

NH₂ silane 1, 2, and 3 are glass plate samples prepared with3-aminopropyltriethoxysilane using the same procedure but on differentdays. Silane 1 was prepared the same day as NH₂ Br 270′, and contactangle measurements were performed at the same time. The differencebetween silane 1, 2, and 3 is also attributed to aging of the glassplates (the time that separates derivatization and contact anglemeasurement, this is also linked to the storage conditions of the glassplates: dry in contact with the air, or kept in solution. Silane 1 wasdried after derivatization, contact angle was measured 1 h later. Silane2 was dried after derivatization, contact angle was measured 10 h later.Silane 3 was kept in toluene after derivatization, and the contact anglewas measured 10 h later. There are no significant differences betweenthe contact angle of NH₂ silane 1 and NH₂ Br 270′.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, genetics, chemistry or related fields are intended tobe within the scope of the following claims.

1. A method of providing complementary nucleic acid comprising: a)providing oligonucleotides, wherein said oligonucleotides are generatedaccording to the method of: A) providing: i) a substrate comprising anarray of oligonucleotides attached to said substrate via anchor moietiesattached to non-cleavable linkers attached to said substrate, whereinsaid anchor moieties comprise the structure —C(X)—C(Y), wherein Xcomprises —OPO₂O—, Y is a nucleophile and wherein said structure is partof a ring moiety; ii) a deprotecting solution; and iii) and a washsolution; B) deprotecting said oligonucleotides with said deprotectingsolution; C) washing said oligonucleotides with said wash solution; andD) cleaving said oligonucleotides at said anchor moieties to providepurified oligonucleotides; b) mixing said oligonucleotides with apolymerase and template strands having a complementary sequence to theoligonucleotide in a polymerization buffer; and c) recovering copies ofcomplementary nucleic acid strands in the duplex with the templatestrand.
 2. A method of providing amplified copies of complementarydeoxyribonucleic acid (DNA) comprising: a) providing oligonucleotides,wherein said oligonucleotides are generated according to the method of:A) providing: i) a substrate comprising an array of oligonucleotidesattached to said substrate via anchor moieties attached to non-cleavablelinkers attached to said substrate, wherein said anchor moietiescomprise the structure —C(X)—C(Y), wherein X comprises —OPO₂O—, Y is anucleophile and wherein said structure is part of a ring moiety; ii) adeprotecting solution; and iii) and a wash solution; B) deprotectingsaid oligonucleotides with said deprotecting solution; C) washing saidoligonucleotides with said wash solution; and D) cleaving saidoligonucleotides at said anchor moieties to provide purifiedoligonucleotides; b) mixing said oligonucleotides with a DNA polymeraseand DNA template strands having a complementary sequence to theoligonucleotide at the 3′-end in a PCR buffer; and c) recoveringamplified copies of complementary DNA strands in the duplex with thetemplate strand.
 3. A method of providing ligated DNA comprising a)providing: i) oligonucleotides obtained according to the method of: A)providing: i) a substrate comprising an array of oligonucleotidesattached to said substrate via anchor moieties attached to non-cleavablelinkers attached to said substrate, wherein said anchor moietiescomprise the structure —C(X)—C(Y), wherein X comprises —OPO₂O—, Y is anucleophile and wherein said structure is part of a ring moiety; ii) adeprotecting solution; and iii) and a wash solution; B) deprotectingsaid oligonucleotides with said deprotecting solution; C) washing saidoligonucleotides with said wash solution; and D) cleaving saidoligonucleotides at said anchor moieties to provide purifiedoligonucleotides; and ii) oligonucleotides which in part form duplexeswith the oligonucleotides of step(i); b) mixing said oligonucleotides of(i) and (ii) with a DNA ligase in a ligation buffer; and c) recoveringligated DNA.